The UDP-sugar hydrolase of Salmonella typhimurium has previously been reported to be located in both the inner and the outer membrane. We have cloned the gene, designated ushB, encoding this enzyme and determined its nucleotide sequence. No significant sequence homology with the periplasmic UDP-sugar hydrolase of Escherichia coli was found at either the DNA or protein level. However, a sequence is detectable, in the E. coli genome, which weakly hybridizes with a specific ushB probe. Polypeptide analysis has allowed the identification of the Salmonella hydrolase which has an Mr of 28,349 as compared to an Mr of 60,767 for the E. coli hydrolase. Most of the protein (approximately 90%) is located in the inner membrane. Two independent membrane fractionation procedures indicate that the remainder may be associated with the outer membrane. The deduced primary structure indicates the presence of an N-terminal signal peptide, although certain features of the region surrounding the putative processing site indicate that processing may be inefficient, or may not occur. Experiments with several inhibitors of signal peptidase function fail to demonstrate the appearance of a precursor form.