Bovine serum was used to identify a recombinant phage clone carrying the Pasteurella haemolytica leukotoxin gene. This fragment produced the 102-kD leukotoxin and several smaller P. haemolytica-specific protein antigens in Escherichia coli. An additional contiguous fragment, containing sequences upstream from the leukotoxin gene. Using these clones, we determined the nucleotide sequence of a 7745-bp region that included four open reading frames: an upstream gene, lktC; the leukotoxin gene, lktA; and two downstream genes, lktB, and lktD. The predicted molecular weights of the proteins encoded by these genes were 19.9, 102, 79.6, and 54.7 kD, respectively. These genes and their predicted proteins were similar in organization and in sequence to the corresponding elements of the gene cluster that encodes an E. coli alpha-hemolysin and its activation and secretion functions. Expression of the leukotoxin was enhanced in E. coli, by fusing the gene to the lac promoter. Under these conditions the leukotoxin was not secreted into the medium, as it is in P. haemolytica. However, in the presence of the alpha-hemolysin genes, the leukotoxin was secreted into the medium, demonstrating functional complementation by the hemolysin secretory system.