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A complete randomized block design was used to compare the binding kinetics of cholera toxin to developing rat enterocyte microvillus membranes prepared from newborn, 2-week-old, 4-week-old, and adult animals. Saturation-binding isotherms were generated on 16 independent samples (four blocks) under steady-state and reversible conditions. Scatchard analyses suggested positive cooperative binding to a single class of receptors, and the isotherms were analyzed by both the Hill-Waud and Michaelis-Menten functions. Receptor density varied significantly with age (P = 0.013). An abrupt rise in receptor density occurred after the neonatal period and normalized in the adult animal. The half-dissociation constant also varied significantly with age (P = 0.019). Microvillus membranes from suckling animals had a slightly higher apparent affinity than those from weaned animals. Neither receptor concentration nor membrane purity confounded these observations. Whereas age-related changes in apparent affinity correlated with cellular responses, changes in receptor density did not. This study suggests that developmental changes in membrane structure which influence binding affinity but not receptor density may, in part, contribute to the increased sensitivity of suckling rats to cholera toxin exposure.