The gonadotropin-releasing hormone (GnRH) waveform arrives at the pituitary gonadotropes via the pituitary portal blood and provides the immediate suprapituitary stimulus to luteinizing hormone (LH) secretion. Despite their importance, nature and influence of the physiological GnRH waveform in vivo have been difficult to study. Recent pharmacological and in vitro studies have suggested the potential importance of the wave contour as a specific and independent factor in the pharmacodynamic effects of GnRH on pituitary gonadotrope LH secretion in vivo, and it has been hypothesized that the steepness of the rising edge of the GnRH wave contour is a specific determinant of pituitary LH secretion. In order to investigate the pharmacodynamic influence of GnRH pulse wave contour on pituitary LH secretion in vivo, variations in plasma LH responses to alterations in GnRH wave contour were measured in chronic ovariectomized, hypothalamopituitary-disconnected sheep undergoing physiological pulsatile GnRH maintenance regimen at a fixed dose (250 ng/pulse) and frequency (interpulse interval 120 min). Variable wave contours were then generated by administration of the same total GnRH pulse dose over various lengths of time from near-instantaneous bolus to increasing lengths of constant-rate infusion time up to 18 min. This model allowed specific examination of pulse wave contour in the absence of concurrent changes in endogenous GnRH or sex steroid secretion and holding constant GnRH pulse dose, frequency, and route of administration. Prolongation of the pulse infusion interval from rapid bolus to 6, 12, and 18 min with a consequent progressive flattening of the GnRH waveform in the bloodstream produced distinctive time course changes (p < 0.0001) with a progressive decrease (p < 0.0001) and delay (p < 0.0001) in peak as well as reduced net (p < 0.0001) LH secretion. These single-dose pharmacodynamic results provide direct support for the hypothesis that the wave contour of a GnRH pulse is a significant determinant of pituitary LH secretion in vivo in sheep. Together with the observed heterogeneity of GnRH patterns in pituitary portal plasma, this suggests the existence of an additional novel mode of hypothalamic regulation of pituitary LH secretion, that of the modulation of the GnRH waveform in the pituitary portal blood.