The gene that encodes the bifunctional short chain isoprenyl diphosphate synthase (idsA) for synthesis of farnesyl diphosphate and geranylgeranyl diphosphate in Methanobacterium thermoautotrophicum, a strict archaebacterial anaerobe, was isolated from a genomic DNA library by colony-lift hybridization and sequenced. Amino acid sequences were obtained for the N-terminus of the enzyme and for internal peptide fragments generated by proteolysis and treatment with cyanogen bromide. Degenerate primers based on the amino acid sequences were used in PCR to synthesize a 220-bp probe from genomic DNA. The probe was radiolabeled and used to isolate idsA. DNA sequencing revealed a 975-bp open reading frame located within an operon. The encoded 325-amino-acid protein contained five conserved regions found in eubacterial and eukaryotic farnesyl diphosphate and geranylgeranyl diphosphate synthases, including aspartate-rich motifs commonly found in prenyltransferases.