The assembly of a polyisoprenoid side chain and its transfer to para-hydroxybenzoate are the first two steps of coenzyme Q biosynthesis. In yeast these reactions are catalyzed by hexaprenyl pyrophosphate synthetase and PHB:polyprenyltransferase, respectively. We have screened nine complementation groups of yeast coenzyme Q mutants for the activities of these two enzymes and found two strains deficient in either activity. The strain deficient in hexaprenyl pyrophosphate synthetase activity, C296-LH3, is complemented by the plasmid pG3/T1. When C296-LH3 was transformed with a shuttle vector containing a 2,187-base pair fragment from the genomic insert of pG3/T1, both glycerol growth and hexaprenyl pyrophosphate synthetase activity were restored. The activity of the latter enzyme was higher than that seen in wild-type yeast. The increase in activity could be attributed to a gene dosage effect of the multi-copy plasmid. A 1,419-base pair open reading frame encoding a 52,560-dalton protein was found on the genomic fragment. The size of the RNA transcript and the location of transcriptional initiation indicate that the entire open reading frame is contained within the mRNA. Comparison of the hexaprenyl pyrophosphate synthetase amino acid sequence with amino acid sequences from the related enzyme farnesyl pyrophosphate synthetase show the presence of three highly conserved domains. Within two of the domains is an aspartate-rich motif found invariantly in the amino acid sequences of farnesyl pyrophosphate synthetase from three species and the hexaprenyl pyrophosphate synthetase amino acid sequence reported here. These aspartic acid motifs may comprise binding sites for the allylic and homoallylic substrates. The hydrophobicity profiles of the hexaprenyl pyrophosphate synthetase sequence and the farnesyl pyrophosphate synthetase sequence from rat appear similar. Furthermore, the hydrophobicity correlation coefficient of the comparison of these two sequences indicate with a high degree of confidence (p less than 0.001) that the two proteins will fold into similar three-dimensional structures.