The origin of hydrogen sulfide in southeastern Montana groundwaters was investigated. Sulfate‐reducing bacteria were detected in 25 of 26 groundwater samples in numbers ranging from 2.0 × 101to greater than 2.4 × 104bacteria per 100 ml. Stable sulfur isotope fractionation studies indicated a biological role in sulfate reduction. However, sulfate‐reducing activity as determined by use of a radioactive sulfur isotope was observed in only 1 of 16 samples. It is postulated that bacterial dissimilatory sulfate reduction is responsible for a major portion of the sulfide produced in these groundwaters and that these bacteria are most likely active in the adsorbed state, possibly in subsurface microzones where environmental conditions are conducive to sulfate reduction.