Dissociation of intact and degraded insulin from hepatocytes in monolayer culture was examined under conditions in which processing of insulin was altered by either temperature or pharmacologic agents. Conditions which increased insulin degradation or processing decreased equilibrium insulin binding whereas those conditions which inhibited processing increased equilibrium binding. The effect of lysosomotropic agents on processing was markedly temperature dependent. Not only was processing increased at higher temperatures (37 degrees C, but the effect of lysosomal inhibitors (chloroquine and methylamine) on insulin processing was abolished at this temperature. The temperature dependency of this effect may explain discrepancies between laboratories on the effect of these inhibitors in hepatocytes.