Cultured GH3 rat pituitary tumor cells which secrete prolactin (PRL) were used to study autoregulation of rat PRL (rPRL) secretion. Acute (1 h) rPRL release by cells in monolayer culture was inhibited by ovine PRL (oPRL; 10-7 and 10-8 M). Intracellular rPRL accumulation was not inhibited by exogenous oPRL. No effect of oPRL was seen during long-term (12 day) incubation of cells with oPRL (10-7 to 10-9 M). oPRL exerts an acute suppressive effect on rPRL release at the cellular level, but does not inhibit rPRL synthesis or long-term release.