Several regulatory peptides, including neuropeptide Y, can release histamine from mast cells. In the present study we investigated which parts of the neuropeptide Y molecule are required to evoke the release of histamine from isolated rat peritoneal mast cells. In addition, we examined whether the histamine release evoked by neuropeptide Y (and by compound 48/80) is sensitive to the G protein inhibitors pertussis toxin and benzalkonium chloride. Neuropeptide Y released histamine in a concentration-dependent manner. Also a neuropeptide Y analog with the center part substituted by 8-aminooctanoic acid, [Aoc2-27]neuropeptide Y, and the cyclic form of the C-terminal hexapeptide, cyclic neuropeptide Y-(31-36), released histamine. The three peptides were equally effective and equally potent. Neuropeptide Y-(1-24)NH2 also released histamine, but its efficacy was low. The rank order of potency of the analogs tested did not agree with that of any of the previously known or postulated neuropeptide Y receptors. Pretreatment of mast cells with pertussis toxin or benzalkonium chloride markedly inhibited the histamine release evoked by neuropeptide Y, [Aoc2-27]neuropeptide Y and compound 48/80. In conclusion, most of the histamine-releasing activity of neuropeptide Y resides in the six C-terminal amino acid residues. The release appears to be G protein-dependent and is probably not receptor mediated.