Betaine lipids are non-phosphorous glycerolipid analogs of phosphatidylcholine. The biosynthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine has previously been studied in phosphate-starved cells of the purple bacterium Rhodobacter sphaeroides, and a genetic approach identified two proteins that are necessary for this process. Here, we show that all reactions of DGTS biosynthesis in R. sphaeroides are attributable to RsBtaA and RsBtaB, as co-expression of the respective genes leads to DGTS formation in Escherichia coli, which normally lacks this lipid. The recombinant RsBtaA protein was membrane-associated and showed S-adenosylmethionine/diacylglycerol 3-amino-3-carboxypropyl transferase activity. RsBtaA directed the transfer of label from 1-[(14)C]S-adenosylmethionine or [(14)C]diacylglycerol at equal rates into the betaine lipid precursor diacylglycerylhomoserine identifying both metabolites as the substrates of the reaction. Comparative analysis of RsBtaA and its bacterial orthologs revealed a motif with similarity to the AdoMet binding pocket of methyltransferases, and allowed the prediction of residues involved in substrate binding.