Objectives were to (1) characterize the relationship of third-ventricle (IIIV) cerebrospinal fluid (CSF) concentrations of growth hormone-releasing hormone (GHRH) with concentrations of GH in the peripheral circulation; and (2) assess the influence of acute administration of appetite-regulating peptides leptin (anti-orexigenic) and neuropeptide Y (NPY; orexigenic) on the release of GHRH. Six mature beef cows fitted with IIIV and jugular vein cannulae were treated intracerebroventricularly with saline, and leptin (600 microg) and NPY (500 microg) in saline, in a replicated 3x3 Latin square design. Third-ventricle CSF and blood were collected 10 min before and continued 220 min after treatments. Mean concentrations of GHRH and frequency of pulses after treatments were 2.2+/-0.13 ng/mL and 1.2+/-0.15 pulses/220 min, respectively. These measures were not influenced by treatments. Concentrations of GHRH in CSF were weakly correlated (r=0.15; P<0.03) with serum concentrations of GH; however, 58% of the GH pulses were preceded by a pulse of GHRH and 90% of the GHRH pulses occurred within 20 min preceding a pulse of GH. Leptin tended (P<0.10) to suppress GH area under the curve (AUC) compared to saline. Concomitantly, NPY tended (P<0.10) to increase GH AUC, which appeared to be a consequence of increased (P<0.05) pulse amplitude. Infusion of NPY also increased (P<0.05) AUC of GHRH relative to saline. No differences were detected among treatments in serum concentrations of insulin-like growth factor-I or its AUC. Sampling CSF from the IIIV appears to be a viable procedure for assessing hypothalamic release of GHRH coincident with anterior pituitary gland secretion of GH in cattle. These data also demonstrate the differential responsiveness of the GH axis to appetite-regulating peptides.