Abstract Vertical distributions of sulfate-reducing bacteria and methane-producing archaea were investigated in the profundal sediment of a freshwater lake using membrane-immobilized small subunit rRNA hybridization with group- and genus-specific oligonucleotide probes. The annual average of the relative abundance of small subunit rRNA hybridized with all probes for sulfate-reducing bacteria to total small subunit rRNA was 2.3% at 0-2 cm and increased with depth up to 22.9% at 8-14 cm where sulfate concentration was less than 10 nmol ml(-1) in interstitial water, suggesting that these bacteria may survive on alternative metabolisms. The signal of probe Dsv687 (the family Desulfovibrionaceae and some Geobacteraceae) was the main factor in this increase. The relative abundance of methane-producing archaea to total small subunit rRNA was highest (7.8%) at 8-14 cm, dominated by the order Methanosarcinales. The metabolic rates measured in the sediments demonstrated that the peaks of sulfate reduction and methane production were separated vertically, and were not linked to their small subunit rRNA distributions. Our data indicate that sulfate-reducing bacteria can coexist with methane-producing archaea from 0 to 20 cm in the freshwater lake sediment.