The effects of pineal indole compounds on the response of the neonatal rat anterior pituitary gland to LH-releasing hormone (LHRH) were studied in organ culture. After 24 h of culture under control conditions, pituitary glands from 5-day-old female rats were routinely incubated for an additional 24 h with the test compounds. Medium LH content was determined by double antibody radioimmunoassay. LHRH (10(-9) M) induced a 10-fold increase in LH levels over control values. Melatonin at a concentration of 10(-9) M significantly reduced the LHRH-stimulated release of LH; maximal suppression to 14% was attained with 10(-8) M melatonin. Similarly, 10(-8) M LHRH caused a 26-fold elevation in medium LH which was suppressed to 62, 55, and 47% by 10(-9), 10(-8), and 10(-7) M melatonin, respectively. In short-term experiments, inhibition was evident within 30 min of treatment. A developmental study of the effect of melatonin on LH release revealed significant inhibition with pituitary glands from rats 2, 5, and 10 days of age but not with glands from animals 21 and 30 days of age. Other pineal indoles were tested for their effects on the LH response to a single dose of LHRH (3 x 10(-10) M). Serotonin at a concentration of 10(-9) M significantly suppressed LH release; maximal reduction to 37-53% occurred with 10(-8) to 10(-6) M serotonin. 5-Methoxytryptamine also produced an inhibition which was significant only at 10(-6) M, the highest concentration tested. N-Acetylserotonin, 5-hydroxyindoleacetic acid, 5-methoxyindoleacetic acid, 5-hydroxytryptophol, and 5-methoxytryptophol showed no consistent inhibitory activity at doses up to 10(-7) M. These findings indicate that melatonin, serotonin, and 5-methoxytryptamine can act directly on the neonatal pituitary gland to suppress LHRH-induced release of LH.