We have previously demonstrated that hypercalcemia in association with a transplantable Leydig cell tumor caused marked alterations in the hypothalamic-pituitary axis of the rat. To determine if the alterations were the result of hypercalcemia rather than other tumor-related factors, we studied the effects of hypercalcemia on the neuroendocrine axis by infusing varying concentrations of CaCl2 into the femoral vein of rats. Three groups of 40 mature male rats each received TRH (10 μg/kg), haloperidol (H; 0.5 mg/kg), or LHRH (10 μg/kg) through an indwelling cannula in the right common carotid artery 30 min after calcium infusion was initiated. Blood samples (100 μl) were drawn through the cannula 30 min before, during and 15, 30, and 45 min after TRH, LHRH, and H. Basal TSH, PRL, and LH levels were suppressed after calcium infusion, and hormonal responses to each of the releasing agents were blunted proportionally to the degree of hypercalcemia achieved. There were significant negative correlations (r = −0.72 to r = −0.83; P < 0.01) between serum calcium levels and TSH responses to TRH, PRL responses to TRH and H, and LH responses to LHRH at the time of administration of the releasing agents. Anterior pituitary hormone secretion of TSH, PRL, and LH was suppressed proportionally to the magnitude of hypercalcemia achieved in the rat. Thus, it appears that serum calcium levels should be taken into account when evaluating the hypothalamicpituitary axis.