The effect in vitro of porcine proinsulin and single component porcine insulin on the epididymal adipose tissue and the isolated diaphragm of the normal rat has been investigated. Both proteins enhance the oxidation of labeled glucose to carbon dioxide and its conversion to fatty acids by adipose tissue as well as glycogen formation from glucose by the hemidiaphragm. Kunitz pancreatic trypsin inhibitor (KPTI) but not the purified Kazal or soybean trypsin inhibitor block these effects of proinsulin on adipose tissue and diaphragm. KPTI has no effect on the response of these tissues to the single component insulin. Anti-insulin serum blocks the response of both adipose tissue and hemidiaphragm to proinsulin and single component insulin. The enhancement of the oxidation of carbon one of glucose to carbon dioxide by adipose tissue in the presence of proinsulin is also blocked by KPTI. It is concluded that porcine proinsulin itself cannot affect the glucose metabolism of adipose tissue and hemidiaphragm of the normal rat. Proinsulin exerts the biologic effect seen in this study because it is converted by an enzyme or group of enzymes present in adipose tissue and diaphragm muscle, to a molecule which is similar to or identical with insulin.