Soil microcosms have been used to demonstrate the ability of indigenous soil thermophiles to degrade effectively a representative alkane (n-hexadecane). A fragment of the alkane mono-oxygenase gene (alkB) was amplified from thermophilic Geobacillus thermoleovorans strain T70 by PCR using degenerate primers. The amplicon demonstrated 96% sequence similarity with the alkB gene from Rhodococcus erythropolis. Critical controls ensured that the positive PCR signal detected was not a result of mesophilic soil organisms. A reverse transcription PCR (RT-PCR) assay was developed to determine if expression of the gene was inducible in the presence of an alkane or constitutively expressed in soil. In the presence of n-hexadecane, expression of the alkane mono-oxygenase gene was induced in pure cultures and soil samples and was dependent on temperature. No positive RT-PCR signal was detected at mesophilic growth temperatures either in pure cultures or in soil microcosms, whereas at 55 degrees C positive RT-PCR signals were obtained for both pure cultures of T70 and soil samples. Many different amplicons of the alkB gene fragment were obtained from the soil used in the microcosms. Thirty cloned fragments yielded 27 different sequences showing 85-96% sequence similarity with the alkB sequence of T70. To establish that the amplified alkB gene sequences from soil were derived from thermophilic geobacilli, additional strains were isolated on a selective medium containing n-hexadecane as sole carbon source. The 16S rRNA gene sequences were determined to identify the 50 isolates obtained (G. thermoleovorans, 27; G. caldoxylosilyticus, 17; G. pallidus, 2; G. toebiii, 1; Geobacillus sp., 3) representing 18 different strains and alkB gene sequences determined and deposited with the European Bioinformatics Institute.