The dum gene of Salmonella typhimurium was originally identified as a gene involved in dUMP synthesis (C. F. Beck et al., J. Bacteriol. 129:305-316, 1977). In the genetic background used in their selection, the joint acquisition of a dcd (dCTP deaminase) and a dum mutation established a condition of thymidine (deoxyuridine) auxotrophy. In this study, we show that dum is identical to pyrH, the gene encoding UMP kinase. The level of UMP kinase activity in the dum mutant was found to be only 30% of that observed for the dum⁺ strain. Thymidine prototrophy was restored to the original dum dcd mutant (KP1361) either by transduction using a pyrH⁺ donor or by complementation with either of two pyrH⁺-carrying plasmids. Thymidine auxotrophy could be reconstructed in the dum⁺ derivative (KP1389) by the introduction of a mutant pyrH allele. To define the minimal mutational complement necessary to produce thymidine auxotrophy in thyA⁺ strains, a dcd::Km null mutation was constructed. In the wild-type background, dcd::Km alone or in combination with a pyrH (dum) mutation did not result in a thymidine requirement. A third mutation, cdd (cytidine-deoxycytidine deaminase), was required together with the dcd and pyrH mutations to impart thymidine auxotrophy.