125I-labeled cholecystokinin (CCK) binding and internalization were studied as a function of temperature in isolated rat pancreatic acini. At 37 degrees C, acini readily bound and degraded 125I-CCK. When labeled hormone binding was inhibited by increasing amounts of unlabeled CCK, competition-inhibition curves were biphasic, consistent with both high- (Kd, 18 pM) and low-affinity (Kd, 13 nM) binding sites. At 4 degrees C, acini bound only one-third as much 125I-CCK and degradation was essentially abolished. At 4 degrees C, CCK competition curves were consistent with a single class of low-affinity binding sites (Kd, 19 nM). Internalization of 125I-CCK was evaluated by three washing procedures utilizing acid, base, and trypsin. All were shown to remove membrane-bound 125I-CCK, and this finding was validated for trypsin by electron microscope autoradiography. After 1 h at 37 degrees C, washing showed 67% of bound 125I-CCK to be internalized and autoradiography showed 54% to be internalized. At 4 degrees C, internalization of bound CCK was greatly reduced but not abolished. When internalization of 125I-CCK was evaluated as a function of the medium concentration of CCK, both high- and low-affinity components were observed. These results suggest that high-affinity CCK binding and CCK internalization are separate temperature-sensitive processes. Moreover, internalization is not uniquely associated with high-affinity binding.