Plasmids that express an enzymatically active beta-galactosidase in an Escherichia coli delta lac strain were isolated from libraries of recombinant plasmids containing Vibrio cholerae chromosomal DNA. Deletion analysis localized the gene responsible for beta-galactosidase activity on a 3.1-kb DNA fragment, and the gene product was identified as a protein of approximately 110 kDa. Primary sequence comparisons indicated that this V. cholerae gene is homologous to the E. coli lacZ gene. In contrast to the lac loci of other bacteria, no gene that could specify a lactose transport system was detected in the vicinity of the V. cholerae lacZ gene, which may account for the inability of this species to use lactose. In V. cholerae, portions of open reading frames encoding proteins homologous to the Alcaligenes eutrophus chrA and E. coli galR gene products were detected upstream and downstream from the lacZ gene, respectively.