The effects of pituitary adenylate cyclase activating polypeptide (PACAP) hybrid, a synthetic antagonist, was investigated on NIH/3T3 cells containing PACAP receptor (R) splice variants (SVs). PACAPhybrid inhibited 125I-PACAP-27 binding to NIH/3T3 cells stably expressing PACAP-R basic, SV-1, SV-2 or SV-3 with an IC50 of 1000 nM. PACAPhybrid antagonized the ability of PACAP-27 to elevate cAMP regardless of the PACAP-R SV used. PACAP was more efficacious at increasing cytosolic Ca2+ in NIH/3T3 cells containing PACAP-R SV-2 than PACAP-R basic, SV-1 or SV-3. PACAPhybrid antagonized the increase in cytosolic Ca2+ caused by PACAP-27 regardless of the PACAP-R SV used. PACAP was more potent at elevating c-fos mRNA using NIH/3T3 cells transfected with PACAP-R SV-2 than PACAP-R basic, SV-1 or SV-3. PACAPhybrid antagonized the increase in c-fos mRNA caused by PACAP-27. These data suggest that PACAPhybrid is a useful PACAP receptor antagonist for PACAP-R SVs.