The structure of rat intestinal cell receptors for Escherichia coli heat-stable enterotoxin (ST) was investigated by affinity cross-linking to 125I-ST and analysis by denaturing gel electrophoresis. Cross-linking of labeled toxin to intestinal membranes and analysis by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed five specifically labeled proteins with molecular masses of 160, 136, 78, 71, and 56 (kilodaltons) kDa. Exhaustive reduction of these samples resulted in a similar pattern of labeling. Affinity-labeled proteins were further analyzed by nonreducing SDS-PAGE, reduction of the resulting separated proteins, and further separation by SDS-PAGE in the presence of beta-mercaptoethanol. Thus, the 160-kDa band on nonreducing gels consisted of two different receptors: a 160-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 78-kDa subunit. Similarly, the 136-kDa band on nonreducing gels consisted of a 136-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 71-kDa subunit. The 78-, 71-, and 56-kDa subunits were not further reducible. These data suggest heterogeneity of the ST receptor subunit structure and organization in rat intestinal epithelia.