Hemolysin BL (HBL), a diarrheal enterotoxin originally isolated from Bacillus cereus strain F837/76, is composed of three antigenically distinct proteins designated B, L1, and L2. All three components are required for biological activity. Here, we report antigenic and physical variations in HBL components produced by other B. cereus isolates. Reactions of partial identity were observed in double gel immunodiffusion assays using antibodies to highly purified B, L1, and L2 components of F837/76 and culture supernatants of strains F837/76 and S1C. Western blot analysis showed that F837/76 produced one 38-kDa B protein, one 38-kDa L1, and one 43-kDa L1 protein. In strain S1C, two B (38 and 42 kDa), two L1 (38 and 41 kDa), and one L1 (43 kDa) proteins were detected. Further Western blot analysis of 127 B. cereus isolates showed that 90 produced one or more of the three HBL components. Approximately half of these 90 isolates (43/90; 48%) produced protein profiles which differed from that of F837/76. A total of four B, two L1, and three L2 component profiles with proteins of different sizes were observed. Individual strains produced various combinations of single or multiple bands of each component. In addition, some strains produced only one or two of the three HBL components. The public health significance of these strains is unknown, as all three components are required for biological activity. The data presented here demonstrates a high degree of heterogeneity in HBL and provide the basis for further studies to characterize the variations in HBL and to determine the role of the variant components in pathogenicity.