In vivo studies have indicated that pancreatic polypeptide induces gallbladder relaxation, whereas motilin initiates contraction of the gallbladder. To determine if these two polypeptides act directly on the gallbladder muscle, their effect on strips of human gallbladder was studied in vitro. Preparations were suspended in an organ bath and the isometric tension recorded. Dose-response curves to cholecystokinin and acetylcholine were first established. The ability of pancreatic polypeptide to cause relaxation under basal conditions and during 50% maximal stimulation by cholecystokinin-octapeptide (2 X 10(-8) M) was assessed on four strips at approximate physiological concentration (300 pmol/liter) and on four additional strips at 10(3) higher concentration. Pancreatic polypeptide did not have any effect on the basal or the cholecystokinin-generated tension at either concentration. The response to motilin was evaluated on four gallbladder strips at concentrations ranging from 10(-11) to 6.7 X 10(-7) M. Although the highest concentration was more than 10(5) greater than levels reported in fasting serum, motilin did not initiate any gallbladder strip contraction. Whereas the preparation was unresponsive to pancreatic polypeptide and motilin, it was capable of contracting in a dose-related fashion to the known agonists cholecystokinin and acetylcholine, and the response was blocked by the respective antagonists dibutyryl cyclic GMP and atropine. It thus seems that pancreatic polypeptide and motilin exert their respective actions as sites remote from the gallbladder without directly affecting gallbladder muscle.