The Ordovician Trenton Group (Sherman Fall and Cobourg formations) and the Lindsay (Collingwood Member) and Blue Mountain formations of southwestern Ontario were examined using Rock-Eval pyrolysis, gas chromatography, gas chromatography-mass spectrometry, and incident-light microscopy to evaluate their paleodepositional environments, thermal maturities, and source rock potential. All units contain sufficient amount of oil-prone (type II), predominantly marine organic matter to be considered as petroleum source rocks. Unstructured bituminite with varying proportions of unicellular alginite are the dominant dispersed organic matter macerals. The bituminite typically occurs in massive to laminated, granular or patchy populations that commonly show microtextural relationships. Persistent inclusions of Leiosphaeridia telalganite demonstrate that planktonic algal debris was a primary organic substrate for blooming microbes. Disseminated coccoidal Gloeocapsomorpha prisca is found in minor amounts, usually in association with common to abundant acritarchs. Zooclasts (chitinozoa, graptolites, scolecodonts) and solid bitumen also are present as maceral inclusions within the bituminite network. The biomarker distributions for all of the studied units are those expected for marine organic matter deposited in a clastic-dominated environment. The extracts are characterized by a smooth n-alkane profile, with low abundance of C (sub 20+) members, typical for marine derived organic matter. Pristane/phytane ratios range from 0.97 to 1.72, indicating dysoxic conditions during deposition. Smooth C 31 -C 35 homohopane profiles, T s /T m ratios (typically above 1.0), and a higher concentration of diasteranes relative to regular steranes all appear to indicate the clay-bearing character of these rocks. The predominance of C 30 hopane over C 29 regular sterane is interpreted to reflect a primary microbial input and extensive reworking of the organic matter. Optical (reflectance, fluorescence) and geochemical (T max , biomarker data) thermal maturity parameters indicate that the Trenton and Blue Mountain strata are within the zone of prolific oil generation throughout the whole area of study. The Collingwood shales are mature with respect to petroleum generation in the eastern part (Toronto area) and only marginally mature in the northern part (Georgian Bay area) of the study area. In general, the biomarker composition of the extracts from all examined units is compatible with that of the oils found in the Trenton reservoirs of southwestern Ontario; however, geochemical and geological evidence suggests that the organic-rich shaly laminae within the Trenton Group are the principal source of these oils. Accumulation of organic carbon in the Ordovician sediments of southern Ontario is suggested to derive from low-energy, normal-marine environments grading from shallow-shelf into deep-shelf and open-basinal settings. The nutrient availability and, consequently, higher bioproductivity, more intense consumption of oxygen, and progressing anoxia, controlled by a low-latitude location, diminished water circulation, stratification of the water column, and a depressed pycnocline resulted in high preservation rates. The amorphous nature of kerogen reflects significant microbial interaction at the water/sediment interface and within the sediments where reducing conditions must have periodically predominated.