Using microsomes prepared from rabbit mammary gland, the dissociation of prolactin (PRL) from its receptor was determined in the presence of peptide hormones or various concentrations of PRL. Among the hormones tested, PRL (ovine, mouse and bullfrog), human growth hormone and human placental lactogen each accelerated the dissociation of PRL in a manner proportional to their receptor-binding activities. Hormone-dependent dissociation was observed at higher concentrations than those at which the binding of PRL was completely inhibited by lactogenic hormones. In the concentration range 0.1 ng/ml-10 micrograms/ml, PRL increased the rate of dissociation in a logarithmic concentration-dependent manner. It was concluded that the dissociation of PRL from its receptor caused by lactogenic hormones is dependent on the hormone concentration. Arrhenius plot analysis revealed that PRL changed the frequency factor for the dissociation reaction. PRL in the medium inhibited the re-association of dissociated PRL. The data also suggested that PRL regulates the rate of dissociation by interacting with the PRL-receptor complex.