In AR 4-2J rat pancreatic acinar cell membranes, receptors for the two pituitary adenylate cyclase-activating peptides (PACAP) PACAP-27 (the short version of PACAP) and PACAP-38 [the long version, with a carboxyl-terminal (residues 28-38) extension] can be subdivided into (a) type A receptors, with high affinity (Kd, 0.3-0.5 nM) for both PACAP-27 and PACAP-38, and (b) type B receptors, with high affinity for PACAP-38 (Kd, 0.3 nM) but low affinity for PACAP-27 (Kd, 20 nM). Determinants of agonist/antagonist activity in 47 PACAP-27 and PACAP-38 analogs (mono- or disubstituted in positions 1, 2, 3, 20, and 21) or amino-terminally shortened were tested by (a) the occupancy of PACAP-A receptors, preferentially labeled with [125I-N-acetyl-His1]PACAP-27, and that of PACAP-A and -B receptors, both labeled with 125I-PACAP-38, and (b) the resulting activation or inhibition of adenylate cyclase. For PACAP-A receptor recognition, deprotonated His1 was a major determinant for PACAP-27 but not PACAP-38; the Kd of 125I-PACAP-27 decreased 2.4-fold at 37 degrees between pH 6.0 and 7.5 and 3.6-fold at 15 degrees, whereas the IC50 of [N-acetyl-His1]PACAP-27 was less affected and that of PACAP(2-27), PACAP(2-38), and PACAP(1-38) was pH independent. In addition, PACAP-A receptors coupled to adenylate cyclase were much more sensitive to PACAP-38 derivatives than to PACAP-27 derivatives; for instance, [D-Phe2]PACAP-38 was a more potent antagonist (Ki, 5 nM) than [D-Phe2]PACAP-27 (Ki, 350 nM), and PACAP(6-38) was a more potent antagonist (Ki, 7 nM) than PACAP(6-27) (Ki, 300 nM). PACAP-B receptors, apart from showing high affinity for PACAP-38, displayed relatively high affinity for amino-terminally shortened PACAP-38 fragments and poor affinity for PACAP-27 and PACAP-27 fragments.