It has been shown that NPY and select C-terminal fragments of NPY that evoke a hypotensive response upon intraarterial administration in the rat also cause mast cell degranulation and histamine release in vitro. Additionally, elevation of plasma histamine levels has been observed concomitant with the hypotensive effect of NPY and various C-terminal fragments. In order to investigate whether the hypotensive response to NPY18-36 is correlated to this observed elevation of histamine in vivo, we sought to characterize the structural requirements for each activity. We conducted a systematic replacement of each amino acid in NPY18-36 by its D-isomer. Additionally, various modifications were made to the N- or C-terminii of NPY18-36. The following rank order of potency was obtained for the hypotensive action of these analogues of NPY18-36 relative to NPY18-36. Only one analogue ([D-Tyr21]NPY18-36) exhibited significantly enhanced potency. Eleven analogues of NPY18-36, ([D-Thr32]-, [D-Arg35]-, [D-Ile31]-, [D-Leu30]-, [D-Tyr27]-, [D-Ser22]-, [D-Tyr36]-, [D-Gln34]-, [D-Asn29]-, [D-Ala23]-, and [D-Arg33]NPY18-36) were equipotent with NPY18-36. Four analogues ([D-His26]-, [D-Ile28]-, and [D-Ala18]NPY18-36 and -NPY18-27) had reduced potency (10-80%) while eight analogues ([D-Arg19]-, [D-Tyr20], [D-Leu24]-, [D-Arg25]-, [Ac-Ala18]-, [Me-Ala18]-, [desamino-Ala18]NPY18-36 and NPY18-36 free acid) failed to produce a significant hypotensive response (less than 10%) at the doses tested. The sensitivity of NPY18-36 to chiral inversion of single residues or other modifications at the N-terminus suggested the presence of a conformationally well defined N-terminal pharmacophore. Additionally, five NPY18-36 analogues were tested for elevation of plasma histamine levels. The rank order of potency ([D-Thr32]NPY18-36 = [D-Tyr21]NPY18-36 much greater than NPY18-36 greater than [D-Ala18]NPY18-36 greater than [Ac-Ala18]NPY18-36) was correlated with each analogue's potency at evoking a hypotensive response. In contrast, NPY1-36 failed to evoke an elevation in plasma histamine levels despite its hypotensive effects. Hence, we conclude that the magnitude of the hypotensive response evoked by an NPY18-36 analogue is primarily a function of its ability to elevate plasma histamine levels. However, the mechanism underlying NPY1-36-evoked hypotension appears to be different.