The purpose of this study was to elucidate the interactions between pituitary adenylate cyclase-activating peptide (PACAP)-(138) and phospholipids in vitro and to determine whether these phenomena modulate, in part, the vasorelaxant effects of the peptide in the intact peripheral microcirculation. We found that the critical micellar concentration of PACAP-(138) was 0.4-0.9 µM. PACAP-(138) significantly increased the surface tension of a dipalmitoylphosphatidylcholine monolayer and underwent conformational transition from predominantly random coil in saline to -helix in the presence of distearoyl-phosphatidylethanolamine-polyethylene glycol (molecular mass of 2,000 Da) sterically stabilized phospholipid micelles (SSM) (P < 0.05). Using intravital microscopy, we found that aqueous PACAP-(138) evoked significant concentration-dependent vasodilation in the intact hamster cheek pouch that was significantly potentiated when PACAP-(138) was associated with SSM (P < 0.05). The vasorelaxant effects of aqueous PACAP-(138) were mediated predominantly by PACAP type 1 (PAC₁) receptors, whereas those of PACAP-(138) in SSM predominantly by PACAP/vasoactive intestinal peptide type 1 and 2 (VPAC₁/VPAC₂) receptors. Collectively, these data indicate that PACAP-(138) self-associates and interacts avidly with phospholipids in vitro and that these phenomena amplify peptide vasoactivity in the intact peripheral microcirculation.