The purpose of this investigation was to elucidate the differences in the nature and time course of changes in in vitro prolactin (PRL) cell activity between blinded and blind/pinealectomized female hamsters. Adult female golden hamsters were either left intact, blinded and sham-pinealectomized (Blind/Sham) or blinded and pinealectomized (Blind/ Pinx). Twelve weeks after surgeries, animals were killed by decapitation and randomized hempituitaries were incubated for a total of either 30, 60, 90, 120, 180 or 240 min. PRL release in vivo, as estimated by monitoring serum titers of immunoreactive PRL (IR-PRL), was markedly reduced in Blind/Sham animals; pinealectomy completely prevented this depression. PRL storage was assessed by measuring total levels (i.e., medium + pituitaries) of IR-PRL in vitro. Total, pituitary and media values of IR-PRL were all significantly depressed in Blind/Sham females. Pinealectomy of blinded animals almost completely prevented these reductions. PRL synthesis, as evaluated by measuring the amount of 3H-leucine incorporated into PRL in vitro, was profoundly reduced in Blind/Sham females. Surprisingly, pinealectomy failed to prevent the blinding-induced decrease in PRL production. From these data, we conclude that in the light-deprived female hamster the pineal gland inhibits PRL storage and release, while the depression in PRL synthesis may be independent of a pineal influence.