The anterior pituitary (AP) gland secretes 6 different hormones. Prolactin (PRL) is secreted at a relatively high level without stimulation by the hypothalamus, while secretion of the others requires the action of stimulatory factors from the hypothalamus. In order to gain an insight into the mechanism underlying the different spontaneous release patterns of these hormones, we investigated their spontaneous release rate after pretreating rat anterior pituitary cells with trypsin. Rat AP cells were cultured on Cytodex microcarrier beads for 4 days and were then superfused with either control medium or medium containing trypsin (0.25%) for 5 min. The subsequent release rates of the AP hormones were monitored. The basal release of PRL was severely reduced to almost undetectable level and began to recover 120 min after the trypsin-pretreatment. Full recovery was attained over the next 100 min and was delayed by treatment with a protein synthesis inhibitor, cycloheximide (7 microM). In the trypsin-pretreated cells, basal release of PRL and growth hormone (GH) was severely reduced, while that of thyroid stimulating hormone (TSH) and adrenocorticotropic hormone (ACTH) was enhanced and luteinizing hormone (LH) and follicle stimulating hormone (FSH) was not markedly affected by the treatment, suggesting that the suppression of PRL release was not caused by nonspecific damage to the cells. Since trypsin does not readily enter cells, the altered secretion of AP hormones seems to be the result of restricted digestion of the external components of the cells. On the bases of these observations, we predicted that the mechanism of spontaneous release of hormones involves trypsin sensitive proteins (TSMP) on the plasma membranes of the anterior pituitary cells.