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An established cellular line (MDCK) derived from canine kidney showed specific binding for prostaglandin E2, both when the determination of specific binding was carried out in cells attached to a semipermeable support and when this binding was determined in suspended cells in a calcium-free medium. Specific binding for tritiated prostaglandin F2 alpha was also observed, and it was inhibited by increasing concentrations of non-radioactive PGF2 alpha in a dose-related pattern. Cross-reactivity between PGE2 binding and PGF2 alpha was present. Arginine-vasopressin induced an increase in the apical-to-basal water flux when applied to the basolateral surface. No change was observed when the hormone was in contact with the apical surface of the monolayer. Prostaglandin E2 also induced an increase in water flux, with a slower time course than that of arginine-vasopressin. The presence of PGE2 inhibited the increase in water flux induced by antidiuretic hormone. The results suggest that MDCK monolayers depict a physiological antagonism between ADH and prostaglandins which is similar to that observed in natural epithelia.