Isolated abdomen and pheromone gland bioassays were utilized to determine the physiological action of the pheromone-biosynthesis-activating neuropeptide (PBAN) in the corn earworm moth Helicoverpa (= Heliothis) zea. An isolated pheromone gland bioassay showed that synthetic PBAN was active at 0.02 pmol, with maximal activity occurring at 0.5 pmol and 60 min of incubation. Second-messenger studies demonstrated that extracellular Ca2+ is necessary for PBAN activity on isolated pheromone glands. The Ca2+ ionophore A23187 stimulated pheromone biosynthesis alone, whereas the Ca2+ channel blockers La3+ and Mn2+ inhibited PBAN activity. However, the organic Ca2+ channel blockers verapamil and nifedipine did not inhibit PBAN activity. Both forskolin and two cAMP analogues stimulated pheromone biosynthesis in the absence of extracellular Ca2+, indicating that Ca2+ may activate an adenylate cyclase. The biogenic amine octopamine did not elicit pheromone production in isolated gland or abdomen bioassays or when injected into intact female moths. Removal of the ventral nerve chord, including the terminal abdominal ganglia in isolated abdomens, did not affect PBAN stimulation of pheromone production. Similar levels of stimulation were found when isolated abdomens were treated with PBAN in scotophase or photophase.