Peptide analogs of growth hormone-releasing hormone (GHRH) can potentially interact with vasoactive intestinal peptide (VIP) receptors (VPAC(1)-R and VPAC(2)-R) because of the structural similarities of these two hormones and their receptors. We synthesized four new analogs related to GHRH (JV-1-50, JV-1-51, JV-1-52, and JV-1-53) with decreased GHRH antagonistic activity and increased VIP antagonistic potency. To characterize various peptide analogs for their antagonistic activity on receptors for GHRH and VIP, we developed assay systems based on superfusion of rat pituitary and pineal cells. Receptor-binding affinities of peptides to the membranes of these cells were also evaluated by radioligand competition assays. Previously reported GHRH antagonists JV-1-36, JV-1-38, and JV-1-42 proved to be selective for GHRH receptors, because they did not influence VIP-stimulated VPAC(2) receptor-dependent prolactin release from pituitary cells or VPAC(1) receptor-dependent cAMP efflux from pinealocytes but strongly inhibited GHRH-stimulated growth hormone (GH) release. Analogs JV-1-50, JV-1-51, and JV-1-52 showed various degrees of VPAC(1)-R and VPAC(2)-R antagonistic potency, although also preserving a substantial GHRH antagonistic effect. Analog JV-1-53 proved to be a highly potent VPAC(1) and VPAC(2) receptor antagonist, devoid of inhibitory effects on GHRH-evoked GH release. The antagonistic activity of these peptide analogs on processes mediated by receptors for GHRH and VIP was consistent with the binding affinity. The analogs with antagonistic effects on different types of receptors expressed on tumor cells could be utilized for the development of new approaches to treatment of various human cancers.