We recently demonstrated that neuropeptide Y (NPY) potentiates LH-releasing hormone (LHRH)-stimulated LH secretion in vivo, and that these actions of NPY are exerted only under the endocrine conditions leading to preovulatory LH surges, viz. the proestrous or estrogen- and progesterone-primed ovariectomized animal. The present experiments tested the hypothesis that NPY's facilitatory actions are exerted directly at the level of the anterior pituitary gland and depend on in vivo exposure of gonadotropes to the preovulatory endocrine milieu. Animals were killed at 1600 h on either metestrus or proestrus. Anterior pituitary glands (APs) were rapidly removed, cut into eighths, and placed into perifusion chambers. APs were perifused with medium 199 for a total of 8 h, and perfusate samples were collected every 5 min. After 30 min of equilibration, APs received hourly pulses of LHRH alone (10(-8) M), NPY alone (10(-6) M), or LHRH plus NPY. Basal runs consisted of perifusion with medium 199 for 8 h with no peptide treatments. Calculations of total hourly LH and FSH responses revealed that whereas LHRH significantly stimulated gonadotropin secretion from both metestrous and proestrous pituitaries, NPY significantly enhanced LHRH-stimulated LH and FSH secretion only from proestrous pituitaries, i.e. from tissue exposed to the endogenous endocrine milieu under which preovulatory gonadotropin surges are generated. NPY had no facilitatory effect on LHRH-induced gonadotropin secretion from metestrous APs. These results are consistent with our previous in vivo findings and demonstrate that the facilitatory actions of NPY on LHRH-stimulated gonadotropin secretion in vitro are limited to the endocrine conditions under which preovulatory gonadotropin surges are generated.