The microorganisms inhabiting a 91°C hot spring in Yellowstone National Park were characterized by sequencing 5S rRNAs isolated from the mixed, natural microflora without cultivation. By comparisons of these sequences with reference sequences, the phylogenetic relationships of the hot spring organisms to better characterized ones were established. Quantitation of the total 5S-sized rRNAs revealed a complex microbial community of three dominant members, a predominant archaebacterium affiliated with the sulfur-metabolizing (dependent) branch of the archaebacteria, and two eubacteria distantly related to Thermus spp. The archaebacterial and the eubacterial 5S rRNAs each constituted about half the examined population.