Phosphatidylcholine is a major component of membranes in most eukaryotes, but it is found only in a small number of bacteria, where it is synthesized by N-methylation of phosphatidylethanolamine. In yeast and other fungi the methylation of phosphatidylethanolamine to phosphatidylcholine proceeds in two steps: the methylation of phosphatidylethanolamine by phosphatidylethanolamine methyltransferase followed by the methylation of monomethylphosphatidylethanolamine by phospholipid methyltransferase. Here we describe the isolation of two allelic phosphatidylcholine-deficient mutants of Rhodobacter sphaeroides which are unable to methylate phosphatidylethanolamine, monomethylphosphatidylethanolamine, or dimethylphosphatidylethanolamine. A DNA fragment containing a gene designated pmtA, which encodes a 22.9-kDa protein, was found to complement both mutants. Expression of this gene in Escherichia coli, which normally lacks phosphatidylcholine or methylated derivatives of phosphatidylethanolamine, resulted in the formation of phosphatidylcholine. A protein extract derived from the E. coli strain expressing the pmtA gene was able to convert phosphatidylethanolamine, mono- and dimethylphosphatidylethanolamine into phosphatidylcholine. Based on these data we conclude that the product of the pmtA gene catalyzes a sequence of three chemically distinct, methylation reactions beginning with phosphatidylethanolamine and leading to the formation of phosphatidylcholine in R. sphaeroides.