The secretory function of the endocrine and exocrine pancreas in the process of cold acclimation has been studied in rats fed at an ambient temperature of 2 degrees C. In anesthetized rats, plasma glucose decreased immediately after cold exposure and increased again toward the basal level after 20 days of continuous cold exposure. Plasma insulin level in these rats also decreased immediately after cold exposure, and the low level was maintained throughout the whole period of cold exposure. In the isolated perfused rat pancreas, the basal insulin output estimated before the stimulation with glucose decreased immediately after the cold exposure and increased gradually during the continuous cold exposure. In these preparations, the initial and the second phases of insulin output were induced by sustaining stimulation with 16.7 mM glucose. Changes in the glucose-induced initial and second phases showed a diphasic pattern during the course of cold exposure. Thus, there was temporary decrease within a week of cold exposure followed by a gradual increase after 3 wk of this condition. The diphasic insulin output in the preparation isolated from rats fed during cold exposure for 6 wk was twice as much as that in the preparation isolated from the rats fed during exposure to 23 degrees C for 5 wk. In the isolated perfused rat pancreas, a monophasic insulin output was induced by sustaining stimulation with 0.1 microM ACh. The monophasic insulin output showed an increasing tendency during cold exposure, but the increase was insignificant because of a larger individual variation.