The objective was to determine whether the suckling-induced delay in return to estrus postpartum could be explained by changes in hypothalamic LHRH content or ability of the pituitary to release LH and FSH in response to LHRH or 59 mM K+ in vitro. In addition, serum concentrations of several other hormones were measured. Nine Holstein cows were suckled ad libitum by two calves and milked by machine twice daily and eight were milked by machine only from calving until slaughter on day 14 postpartum. On day 13 postpartum, blood was collected at 15-min intervals from 0815 to 1200 hr and from 2015 to 2400 hours. Suckled cows had lower (P less than .05) mean serum LH concentrations on day 13 postpartum than did nonsuckled controls. This decrease resulted from a 60% reduction in frequency and a 40% reduction in amplitude of episodic LH peaks. Suckling did not affect body weight change postpartum or serum concentrations of progesterone, estradiol-17 beta, total glucocorticoids, prolactin or FSH during the first 14 days postpartum. The suckling-induced decrease in serum LH was not reflected by a reduction in hypothalamic LHRH or pituitary LH on day 14 postpartum. However, pituitary explants from suckled cows on day 14 postpartum secreted 50% less (P less than .01) LH in response to LHRH (25 ng/ml for 30 min) or K+ (59 mM for 30 min) in vitro than did those from nonsuckled cows. Secretion of FSH was increased 20-fold by LHRH and K+ in vitro, but differences due to suckling treatment were not significant. Decreased frequency and amplitude of episodic LH secretion in vivo and reduced capacity of pituitaries to respond to LHRH may be the cause of suckling-induced inhibition of postpartum ovulation in cattle.