The CobIII region of the cobalamin (CBL) biosynthetic (cob) operon of Salmonella typhimurium encodes functions necessary for the synthesis of the nucleotide loop of CBL and comprises three genes, designated cobU, cobS, and cobT (26). Complementation studies identified two classes of CobIII mutants: (i) 34 mutants were complemented by a plasmid carrying the cobU+ gene, and (ii) 27 mutants were complemented by a plasmid carrying the cobS+ gene; none of the mutants tested was complemented by the cobT+ clone, a result suggesting that no cobT mutations were isolated. These data were consistent with those of complementation studies done with F' cobUST plasmids, which also suggested that the CobIII region comprises two complementation groups. A plasmid carrying cobUS+ was sufficient to complement a deletion of the entire CobIII region, a result suggesting that CobT was not required for CBL biosynthesis. Nutritional studies done with synthetic putative intermediates of the CobIII pathway were performed to further classify cobIII mutants. A subset of cobU mutants were found to be responsive to exogenous dicyano-cobinamide-GDP, while cobS mutants were found to be responsive only to CBL. These results are consistent with the adenosyl-cobinamide kinase-GTP:adenosyl-cobinamide-phosphate guanylyltransferase and CBL synthase activities proposed for CobU and CobS, respectively. The cobIII genes under the control of the T7 promoter were overexpressed, and the resulting polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three polypeptides with apparent molecular masses of 22, 26 and 39 kDa, consistent with the predicted masses for CobU, CobS, and CobT, respectively, were detected.