The carbon isotopic composition of organic matter is a powerful tool in the study of organic carbon burial and pCO2 through the geologic record. However, the organic-matter source can affect total organic carbon δ13C values and may complicate interpretation of environmental conditions. We examined the influence of organic-matter source variations on a late Middle Ordovician positive δ13C excursion in strata of the United States midcontinent. In a core from Iowa, the magnitude of the excursion recorded by carbonate carbon is ∼2.5‰, whereas the excursion recorded by total organic carbon is nearly 8‰. Although this difference could arise from a profound decrease in surface-water CO2 concentrations, association of the organic-walled microfossil Gloeocapsomorpha prisca with 13C-enriched total organic carbon (TOC) in this interval suggests that the excursion could also record a shift from normal-marine to G. prisca–dominated organic matter. Carbon isotope analyses of individual biomarkers indicate that both of these explanations are required to explain the data. Compounds inferred to be of G. prisca origin are enriched in 13C by 7‰ relative to compounds derived from other algal sources. Furthermore, compounds exclusively from either G. prisca or other sources exhibit a positive shift of only 3.5‰. Thus, the large excursion recorded by TOC reflects, in part, a change in the proportional contribution of G. prisca to TOC. That all compounds exhibit a positive carbon isotopic shift indicates that, independent of contributions from G. prisca, a positive organic δ13C excursion occurred and records a change in environmental conditions such as CO2(aq) concentrations. The 3.5‰ shift is similar in magnitude to that observed for TOC in correlative units from Pennsylvania, suggesting that this event has broad geographic significance. This work illustrates that organic-matter source variations as well as environmental conditions govern the TOC δ13C values and shows how compound-specific isotope analyses can resolve these multiple controls.