1. The mediators of nonadrenergic, noncholinergic (NANC) inhibitory responses in longitudinal muscle of rat distal colon were studied. 2. An antagonist of pituitary adenylate cyclase activating peptide (PACAP) receptors, PACAP6-38, concentration-dependently inhibited the rapid relaxation of the longitudinal muscle induced by electrical field stimulation (EFS), resulting in a maximal inhibition of 47% at 3 microM. 3. PACAP6-38 inhibited the relaxation by 75% in the presence of the vasoactive intestinal peptide (VIP) receptor antagonist, VIP10-28 at 3 microM, which inhibited the relaxation by 44%. 4. An antagonist of large conductance Ca(2+)-activated K+ channels, charybdotoxin, concentration-dependently inhibited the rapid relaxation of the longitudinal muscle, resulting in a maximal inhibition of 58% at 100 nM. 5. An antagonist of small conductance Ca(2+)-activated K+ channels, apamin, concentration-dependently inhibited the relaxation (58% at 1 microM). 6. Treatment with both K+ channel antagonists resulted in 84% inhibition of the EFS-induced relaxation, which is comparable to the extent of inhibition induced by PACAP6-38 plus VIP10-28. 7. The inhibitory effect of VIP10-28 and of apamin, but not of charybdotoxin was additive: the same applied to PACAP6-38 and charybdotoxin, but not apamin. 8. Exogenously added VIP (100 nM 1 microM) induced a slow gradual relaxation of the longitudinal muscle. Charybdotoxin, but not apamin significantly inhibited the VIP-induced relaxation VIP10-28, but not PACAP6-38 selectively inhibited the VIP-induced relaxation. 9. Exogenously added PACAP (10-100 nM) also induced slow relaxation. Apamin and to a lesser extent, charybdotoxin, inhibited the PACAP-induced relaxation. PACAP6-38, but not VIP10-28 selectively inhibited the PACAP-induced relaxation. 10. Apamin at 100 nM inhibited inhibitory junction potentials (i.j.ps) induced by a single pulse of EFS Apamin also inhibited a rapid phase, but not a delayed phase of i.j.ps induced by two pulses at 10 Hz. VIP10-28 did not inhibit i.j.ps induced by a single pulse, but significantly inhibited the delayed phase at two pulses. A combination of apamin and VIP10-28 abolished the i.j.ps induced by two pulses. 11. Both VIP and PACAP induced slow hyperpolarization of the cell membrane of the longitudinal muscle. Apamin inhibited the PACAP-, but not VIP-induced hyperpolarization. 12. From these findings it is suggested that VIP and PACAP are involved in NANC inhibitory responses of longitudinal muscle of the rat distal colon via activation of charybdotoxin- and apamin-sensitive K+ channels, respectively.