This paper presents definitive results which establishes a direct gene-protein product relationship between the pgsA gene and the phosphatidylglycerophosphate synthase of Escherichia coli. The predicted protein sequence derived from the determined DNA sequence of pgsA is in close agreement with the amino acid composition and partially determined amino acid sequence of the purified enzyme. The purified synthase has the same apparent molecular mass as the gene product made by a plasmid-directed transcription-translation system. The plasmid-borne copy of the pgsA gene is also capable of expressing enzymatically active synthase in vitro. The DNA sequence analysis has established the exact linear relationship between the uvrC, pgsA, and glyW loci and revealed that these three genes are transcribed in the same direction. The terminal coding regions of these three genes also share common sequences with transcriptional regulatory elements for the adjacent genes.