Transient removal of dopamine (DA) potentiates the prolactin (PRL) releasing action of thyrotropin releasing hormone (TRH) in cultured lactotrophs. We have determined if this potentiating action on PRL release is related to the intracellular processing of PRL by studying this phenomenon in cells in which PRL was pulse-labeled at varying times. Anterior pituitaries from ovariectomized estradiol-treated rats were dispersed and cultured on plastic coverslips. Cells were incubated for 24 h in media containing 500 nM DA. Cells were exposed to three consecutive 20-min incubation periods. During the first period all cells were maintained in 500 nM DA, during the second period half of the cells were removed from DA inhibition and during the third period DA inhibition was restored and the cells challenged with 100 nM TRH. In experiments involving labeling of PRL, cells were incubated in leucine-free media containing 100 µCi/ml [3H] leucine for 1 h prior to treatment, for 1 h starting 5 h prior to treatment or for 16 h starting 24 h prior to treatment. Total PRL (by radioimmunoassay) and radiolabeled PRL (by liquid scintillation after electrophoretic isolation) were determined both in media and cells. The withdrawal of DA alone resulted in a 2-fold stimulation of total PRL release. The efficacy of TRH to release total PRL was also increased 2- to 4-fold after transient DA withdrawal. Whereas release of 1-hour-old labeled PRL was not affected by DA withdrawal, 5-hour-old and 8- to 24-hour-old [3H] PRLs were stimulated 2.6- and 2-fold, respectively. TRH stimulation of 5-hour-old [3H] PRL release was potentiated 6-fold (from 150 to 900% over the basal) by transient removal of DA. On the other hand, TRH stimulation of 8- to 24-hour-old [3H] PRL release (180% over the basal) was not affected by the prior escape from the action of DA. TRH did not affect the release of 1-hour-old [3H] PRL either under constant or after the transient removal of DA. These results suggest that the regulated release of PRL is highly dependent on the age of the hormone. The potentiation of TRH-induced PRL release evoked by DA removal was only observed with hormone synthesized from 4 to 5 h earlier. PRL synthesized up to 1 h before was constitutively released and neither DA withdrawal nor addition of TRH affected its secretion in spite of the observation that its relative rate of basal release was not different from that of 4- to 5-hour-old PRL. PRL synthesized more than 8 h earlier was regulated independently by TRH and DA, however, transient removal of DA had no effect on potentiation of the TRH response. Therefore PRL stored for prolonged periods of time cannot be coupled to the biochemical events mediating the potentiating effect of transient DA removal on the action of TRH