N-terminally shortened analogues of PACAP(1-27) and PACAP(1-38), and analogues modified in position 1,2 or 3 were compared for their ability to interact with PACAP receptors and to activate or inhibit adenylate cyclase in rat brain hippocampus membranes. In the PACAP(1-27) series, deletion of the first two amino acids decreased the potency 3000-fold. PACAP fragments (3-27) to (9-27) were inactive on the enzyme. N-terminally shortened PACAP(1-38) analogues showed a similar profile but were 70 to 300-fold more potent than their PACAP(1-27) equivalents. PACAP(6-27) and PACAP(6-38) were competitive inhibitors of the PACAP(1-27) stimulated enzyme. The Kd values of PACAP(6-27) and PACAP(6-38) were of 1000 and 2 nM respectively. Surprisingly, the Kd values of PACAP(6-31) and (6-35), that were also unable to stimulate adenylate cyclase activity, were of 3 and 300 nM respectively. Replacement of His1 by Phe1 in PACAP(1-27) reduced the potency 600-fold. Replacement of Ser2 by Ala2 in PACAP(1-27) and PACAP(1-38) was of little consequence. Substitution of Ser2 by Phe2, DPhe2, Arg2 or DArg2 reduced 60 to 1000-fold the PACAP(1-27) potency but only 7 to 30-fold the PACAP(1-38) potency. Phe2 derivatives were inactive on the enzyme. Replacement of Asp3 by Asn reduced 4000-fold the PACAP(1-27) potency.(ABSTRACT TRUNCATED AT 250 WORDS)