We studied the basal and thyrotropin-releasing hormone (TRH)-stimulated in vitro release of newly synthesized and mature prolactin (PRL) by lactating rat pituitary fragments. Newly synthesized PRL was defined as that resulting from either in vivo pulse labelling with [3H]-leucine (injected i.v. 10 min before removing the pituitary glands for in vitro incubation); or in vitro labelled by a 5-min preincubation period of adenohypophyseal fragments. Mature PRL was defined as that labelled in vivo 8 h before incubation. Medium PRL was quantified by polyacrylamide-gel-electrophoresis densitometric and liquid scintillation techniques. Under basal conditions, both total (unlabelled) and 8-hour labelled (mature) PRLs showed an in vitro release pattern characterized by an initial period of faster and higher secretion that lasted for 30-60 min followed by a lower secretion rate that extended from 60 to 240 min of incubation. Secretion of newly synthesized PRLs was detected since the first 30 min, but did not reach maximum levels until 90-120 min of incubation. Accordingly, PRL release during the first 30-60 min of incubation consisted primarily of mature PRL, whereas secretion during the rest of incubation included the release of newly synthesized hormone. These results confirm previous data on the sequential, rather than preferential, release of different age PRLs. TRH (0.35 microM) stimulated (about 30% above control) the release of both total and mature PRLs. No effect could be detected upon the release of in vivo labelled new PRL, while the same dose of TRH strongly stimulated (about 100% above control) the release of in vitro labelled new PRL.(ABSTRACT TRUNCATED AT 250 WORDS)