Hemolysin BL, which is composed of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal food poisoning syndrome caused by strains of Bacillus cereus. To further characterize the toxin, we sought to clone and sequence the genes encoding the L1 and L2 proteins. A genomic library was screened with polyclonal antibody to the L1 and L2 proteins to identify recombinant clones containing the genes. Five clones reacted with the antibody to L2, but none reacted with the antibody to L1. Southern hybridization analysis with oligonucleotide probes designed from the N-terminal amino acid sequences of the L1 and L2 proteins, in conjunction with immunoblot and nucleotide sequence analysis, revealed that the recombinant plasmid from one of the clones contained two genes, hblC and hblD, which encode L2 and L1, respectively. The two genes are arranged in tandem and are separated by only 37 bases. The gene which encodes the B component of hemolysin BL (hblA) is located immediately downstream from the gene encoding the L1 protein. Northern blot analysis of B. cereus RNA showed a 5.5-kb transcript which hybridized with DNA fragments internal to, or including a portion of, the coding sequences of the B, L1, and L2 genes, suggesting that the clustered genes which encode the components of hemolysin BL are cotranscribed and constitute an operon.