A sulfur isotope investigation of six copper sulfide occurrences, in flat-lying Proterozoic sediments at Pernatty Lagoon, South Australia, was undertaken to understand further their genesis and to apply the knowledge gained towards locating further ore bodies. Geological, mineralogical, and delta S 34 studies point to three distinct periods of mineralization and suggest that the bulk of the sulfides in the six areas examined have precipitated from sulfide of biological origin.Mineralization in dolomitic shale at the Sweet Nell and Gully areas is considered to be the oldest and to have formed syngenetically (delta S 34 avg = + 17.4 per mil, Sigma = 10.3 per mil, and delta S 34 avg = + 19.0 per mil, Sigma = 16.1 per mil respectively). The most common copper sulfide minerals present in these two deposits are chalcopyrite and bornite. In the other areas the major copper sulfides are digenite and djurleite which are associated mainly with the Pandurra quartzite and the Whyalla sandstone and were emplaced after lithification of the sediments. The latest mineralization is shown in the East Lagoon and Mystery areas and represents near-surface sulfide mineralization formed under arid conditions, similar to the present-day climate of the area (average delta S 34 value for East Lagoon sulfides= -1.2 per mil, Sigma =11.8 per mil). The mineralization postdates the deposition of the overlying Tregolana shale and the closing-off of Gunyah Lake. The sulfides of the Main and House workings are all enriched in the light isotope S 32 and were formed in a more open (to sulfate) system than the other areas (average delta S 34 = -4.6 per mil, Sigma = 3.7 per mil and -7.6 per mil, Sigma = 4.6 per mil respectively).The restricted environment, indicated by the delta S 34 values for the East Lagoon sulfides, is shown to be related to water movement and metal sulfide precipitation, which occurred mainly along the quartzite-sandstone boundary.