To cope with osmotic stress, Sinorhizobium meliloti accumulates organic compatible solutes such as glutamate, trehalose, N-acetylglutaminylglutamine amide, and the most potent osmoprotectant glycine betaine. In order to study the regulation of the glycine betaine biosynthetic pathway, a genetic and molecular analysis was performed. We have selected a Tn5 mutant of S. meliloti which was deficient in choline dehydrogenase activity. The mutation was complemented using a genomic bank of S. meliloti. Subcloning and DNA sequencing of a 8–6 kb region from the complemented plasmid showed four open reading frames with an original structural organization of the bet locus compared to that described in E. coli. (i) The betB and the betA genes which encode a glycine betaine aldehyde dehydrogenase, and a choline dehydrogenase, respectively, are separated from the betI gene (regulatory protein) by an additional gene named betC. The BetC protein shares about 30% identity with various sulphatases and is involved in the conversion of choline-O-sulphate into choline. Choline-O-sulphate is used as an osmoprotectant, or as a carbon or sulphur source and this utilization is dependent on a functional bet locus. (ii) No sequence homologous to betT (encoding a high-affinity choline transport system in E. coli) was found in the vicinity of the bet locus. (iii) The betB and the betA genes, as well as the betI and the betC genes are, respectively, separated by 211 and 167 bp sequences containing inverted repeats. Southern blot analysis indicated that the bet locus is located on the chromosome, and not on the megaplasmids.