Boiling Springs Lake is an approximately 12,000 m(2), 55 degrees C, pH 2 thermal feature located in Lassen Volcanic National Park in northern California, USA. We assessed the microbial diversity in the lake by analyzing approximately 500 sequences from clone libraries constructed using three different primer sets targeted at 16S rRNA genes and one targeted at 18S rRNA genes. We assessed the stability of the microbial community by constructing terminal restriction fragment length polymorphism (T-RFLP) profiles using DNA extracts collected in four separate years over a 7-year period. The four most prevalent phylotypes in the clone libraries shared an average approximately 85% sequence identity with their closest cultured relatives, and three fourths of the prokaryotic sequences shared less than 91% identity. Phylogenetic analyses revealed novel lineages devoid of cultivated representatives in the Bacterial and Archaeal domains. Many detected phylotypes were related to taxonomically diverse genera previously associated with high-temperature environments, while others were related to diverse Proteobacteria and Firmicutes that would not be expected to grow within BSL conditions. All of the 18S rRNA sequences most closely matched fungi in the phyla Ascomycota and Basidiomycota (91-99% identity). T-RFLP detected fragments corresponding to the most prevalent phylotypes detected in 16S rRNA gene libraries. The T-RFLPs from separate years were similar, and the water-derived T-RFLPs were similar to the sediment-derived (average pairwise Sorenson's similarity index of 0.74, and 0.78, respectively). Collectively, these results indicate that a stable community of diverse novel microorganisms exists in Boiling Springs Lake.