As a first step towards the elucidation of the molecular mechanisms responsible for the utilization of choline and glycine betaine (betaine) either as carbon and nitrogen sources or as osmoprotectants in Sinorhizobium meliloti, we selected a Tn5 mutant, LTS23-1020, which failed to grow on choline but grew on betaine. The mutant was deficient in choline dehydrogenase (CDH) activity, failed to oxidize [methyl-14C]choline to [methyl-14C]betaine, and did not use choline, but still used betaine, as an osmoprotectant. The Tn5 mutation in LTS23-1020 was complemented by plasmid pCHO34, isolated from a genomic bank of S. meliloti 102F34. Subcloning and DNA sequencing showed that pCHO34 harbours two ORFs which showed 60% and 57% identity with the Escherichia coli betB gene encoding betaine-aldehyde dehydrogenase (BADH) and betA gene encoding CDH, respectively. In addition to the homology with E. coli genes, the deduced sequence of the sinorhizobial BADH protein displays consensus sequences also found in plant BADHs. The deduced sequence of the sinorhizobial CDH protein shares only 21% identical residues with choline oxidase from Arthrobacter globiformis. The structural organization of the betBA genes in S. meliloti differs from that described in E. coli: (i) the two ORFs are separated by a 210 bp sequence containing inverted repeats resembling a putative rho-independent transcription terminator, and (ii) no sequence homologous to betT (high-affinity choline transport system) or betI (regulator) was found in the vicinity of the sinorhizobial betBA genes. Evidence is also presented that the S. meliloti betBA genes are not located on the megaplasmids.