Samples of short pink-grayish filaments were collected from a hot spring in the Hengill area in southwestern Iceland at 85-88 degrees C, pH 6.9 and 1.7 mg/L sulfide. The species composition was studied by cloning and sequencing small subunit rRNA genes obtained by PCR amplifications from mat DNA. Using 98% sequence similarity as a cutoff value, a total of 5 bacterial operational taxonomic units (OTUs) and 6 archaeal OTUs were detected among 68 bacterial clones and 97 archaeal clones. Database matching showed that 80.5% of the archaeal sequences were 99% similar to Pyrobaculum islandicum and 14.5% were closest to the Korarchaeota clone sequence SRI306. About 87% of the bacterial sequences had the closest database match (99%) to the clone sequence SRI48 but were also found to be 99% identical with hydrogen-oxidizing strains previously isolated in this laboratory from hot springs in the same region. Out of 7 Thermus sequences, 4 were 100% identical to T. scotoductus NMX2 A.1 but 3 represented a new uncultivated Thermus species. Four different media, varying in organic nutrients and phosphate composition were used to isolate 81 aerobic thermophilic heterotrophs. Four isolates were Bacillus spp; but out of 77 Thermus isolates, 42 belonged to T. scotoductus and 35 to T. brockianus. T. scotoductus seemed to be preferably isolated on media low in nutrients and phosphate, whereas for T. brockianus it was the opposite. The T. scotoductus clones and isolates had 99-100% sequence similarity to each other. No T. brockianus sequences were found in the bacterial clone library.