Studies on hormone-receptor interactions generally assume that the formation of a hormone-receptor complex is a reversible process. This assumption has been examined directly in three experiments using liver membrane receptor preparations from pregnant rats and ovine PRL (oPRL). In Exp 1, Receptors were preincubated with a range of concentrations of oPRL at 23 C for periods up to 60 min, washed thereafter to remove free oPRL, and subsequently incubated with [125I]iodo-oPRL (23 C) to determine specific binding. Preincubation of receptors (0.25 mg membrane protein) with oPRL (5 ng) for periods as brief as 10 min reduced subsequent binding of [125I]iodo-oPRL to receptor, suggesting incomplete dissociation of oPRL even after 30 h. In Exp 2 after preincubation for 30 min with oPRL and subsequent incubation with [125I]iodo-oPRL for 19 h, membranes were washed, and the dissociation (23 or 37 C) of [125I]iodo-oPRL from the hormone-receptor complex in the presence or absence of 1000 ng oPRL was studied. After 48 h, only 35-50% of the [125I]iodo-oPRL dissociated from the hormone-receptor complex even in the presence or excess oPRL, indicating a heterogeneity of binding sites (i.e. 50-65% irreversible; 35-50% reversible). When pregnant rat serum was used in place of oPRL or when rabbit mammary glands were used instead of rat livers to prepare receptor preparations, results were similar to those described above, except for the nearly complete dissociation (90%) obtained at 37 C using rabbit mammary gland receptors. In Exp 3 after incubation (10 min, 2 h, or 15 h) of rat liver receptors with [125I]iodo-oPRL plus various amounts of oPRL, the hormone-receptor complex could be completely dissociated with 5 M MgCl2, restoring binding affinity and capacity of receptor to their original values. Labeled oPRL dissociated by MgCl2 treatment from such a complex is capable of binding to fresh receptor. These data strongly suggest that the PRL-receptor interaction, particularly the rat liver receptor interaction with PRL under usual in vitro conditions, is not reversible to a significant degree. This is not due to hormone or receptor damage but to a significant number of binding sites (50-65%) in the receptor preparation which are not reversible except under extreme conditions.