The antigenic relationships between heat-labile enterotoxin (LT) produced by a human strain of enterotoxigenic Escherichia coli (strain 286C2) and cholera toxin (CT) were examined by using antisera raised against LT and CT and specific antisera prepared against each subunit of both enterotoxins. Double immunodiffusion analysis revealed reactions of partial identity between the A subunits of LT and CT, as well as between the B subunits. Rabbit antisera raised against LT subunit A reacted with only subunit A, whereas rabbits immunized with LT subunit B produced antibodies which reacted with only subunit B. A high degree of CT neutralization was observed with antisera raised against LT. Data from neutralization assays with specific antisera to each enterotoxin showed that LT was more effectively neutralized by homologous anti-LT than CT (3.7-fold); however, anti-CT was only slightly more effective in neutralization of homologous CT compared with LT (1.9-fold). In contrast, antisera raised against the B subunit of CT (choleragenoid) exhibited significantly higher neutralization activity against CT than LT (5.8-fold); however, the amount of CT neutralized by anticholeragenoid was less (4.1-fold) than anti-CT. These results suggested that anti-CT serum contained neutralizing antibodies reactive with a shared determinant formed by interaction of the A and B subunits, whereas anti-LT and anti-choleragenoid sera did not. Sensitive solid-phase radioimmunoassays were developed to examine the affinity and degree of specificity involved in homologous and heterologous antigen-antibody interactions between LT, CT, their subunits, and specific antibodies. Only unlabeled LT competed with radiolabeled LT in polystyrene tubes coated with anti-LT, and only unlabeled CT competed with radiolabeled CT in tubes coated with anti-CT. However, when radiolabeled CT was incubated in tubes coated with anti-LT, competitive inhibition responses were observed with both unlabeled toxins. When radiolabeled LT was incubated with tubes coated with anti-CT, competitive inhibition responses were observed with both unlabeled toxins. Similar competitive inhibition responses were observed with the A subunits of LT and CT and with the B subunits using antisera specific for the subunits of each enterotoxin. Double immunodiffusion analysis and radioimmunoassay data supported the presence of unique and shared immunodeterminants in each subunit.