We have recently reported oral and parenteral bioactivity for a new GH-releasing peptide, hexarelin. In the present study, we have examined the neuroendocrine mechanism by which hexarelin and GHRP-6, two GH-releasing peptides, mediate their actions. Although previous studies have looked at the role of growth hormone-releasing hormone (GHRH) and somatostatin in regulating the action of GHRP-6 in culture and in stressed animals, our study looked at the role of both somatostatin and GHRH in regulating the action of hexarelin as well as GHRP-6 in conscious and freely-moving, nonstressed rats. Adult male rats, prepared with indwelling jugular catheters, were pretreated i.v. with either control antiserum (CTLas), growth hormone-releasing hormone antiserum (GHRHas), somatostatin antiserum (SSas), or both GHRHas and SSas. Animals were then treated i.v. with 25 µg/kg of either hexarelin or GHRP-6 4 h after i.v. antisera pretreatment. Blood samples were collected every 20 min for the 3 h prior to peptide treatment and at 5, 10, 15, 20, 40 and 60 min following hexarelin or GHRP-6 injection. The peak plasma GH responses in rats pretreated with CTLas were 552 ± 125 ng/ml following hexarelin administration and 386 ± 132ng/ml following GHRP-6 administration. Rats pretreated with SSas exhibited peak GH responses following hexarelin or GHRP-6 of 702 ± 115 and 312 ± 42 ng/ml, respectively. These plasma GH responses were similar to those observed in the CTLas-pretreated animals. Hexarelin injection resulted in a peak GH response of 372 ± 106 ng/ml in GHRHas-pretreated rats, while GHRP-6 injection resulted in a peak response of 135 ± 40 ng/ml. Rats pretreated with both GHRHas and SSas responded to hexarelin administration with a GH peak of 322 ± 66 ng/ml and to GHRP-6 with a response of 134 ± 39 ng/ml. GH responses noted in these animals were similar to the responses noted in GHRHas-pretreated rats. The GH responses elicited following either GHRHas or both GHRHas and SSas pretreatment were significantly lower than those responses observed in rats pretreated with either CTLas or SSas alone. Repeated measures analysis of the variance of peak plasma GH responses as well as of the area under the GH response curves allowed us to reach the following conclusions: (1) hexarelin was more effective in eliciting GH release than was GHRP-6 at the dose tested; (2) GHRHas inhibited the GH response to both hexarelin and GHRP-6, and (3) SSas pretreatment did not affect the GH response to the GH-releasing peptides. Therefore, we conclude that these two GH-releasing peptides elicit GH release via a GHRH-dependent pathway. In addition, because both hexarelin and GHRP-6 are bioactive regardless of circulating somatostatin tone, these GH-releasing peptides must also act to suppress somatostatin release from the hypothalamus or somatostatin effectiveness at the pituitary.