We have recently demonstrated that GnRH-stimulated gonadotropin secretion is augmented by coadministration with neuropeptide-Y (NPY) in anterior pituitaries removed in the afternoon from proestrous, but not metestrous, rats. To test the hypothesis that these effects of NPY are due to an interaction with progesterone (P4), we conducted another cycle stage experiment using NPY, adding an in vitro treatment with P4. Pituitaries were taken from rats at 0900 h (before the rise of P4 on proestrus) on proestrus or metestrus and were perifused for 8 h, with and without GnRH pulses. P4 was given continuously in half of each of the basal or GnRH-stimulated perifusions. Pulsatile NPY was administered in half of the basal or GnRH-stimulated runs in the presence or absence of P4. P4 alone had a stimulatory effect on GnRH-induced LH secretion only on the day of metestrus. P4 did not confer responsiveness to NPY stimulation on either day. Unexpectedly, NPY reversed the P4 augmentation of GnRH-stimulated LH release on metestrus. With respect to FSH secretion, NPY or P4 alone had a striking suppressive effect on GnRH-stimulated FSH secretion rates from pituitaries of metestrous, but not proestrous, donors. These data suggest that the previous endogenous endocrine environment may be crucial in determining the divergent and interrelated effects of P4 and NPY on gonadotropin secretion. In particular, the metestrous environment appears to promote a suppressive role for NPY.