In order to elucidate the action of prolactin (PRL) on the dissociation of PRL from the receptor, Triton-solubilized and concanavalin A-purified rabbit mammary gland PRL receptor was immobilized onto a nitrocellulose membrane, and the dissociation of PRL was determined in the presence of PRL. PRL accelerated the dissociation of PRL in a concentration dependent manner for the first 3 h of incubation. The effects of PRL on dissociation were independent of the occupancy of PRL at the zero-time dissociation. In particular, PRL-dependency was influenced by the PRL-binding capacity of the membrane. The dissociation reaction was PRL-independent in the low-binding capacity membrane, indicating that PRL itself had no accelerating actions. With greater binding capacity, the dissociation reaction was seen to become PRL-dependent. It was concluded that PRL accelerated the dissociation of PRL through the formation of PRL-receptor complex when the receptor was located closely to the neighboring receptors.