An endogenous protein of human cells pp60c-src, which is closely related to the product of the transforming gene of Rous sarcoma virus, pp60v-src, has been quantitated by measuring its enzymatic activity in an immunoglobulin G protein kinase assay. The influence of normal developmental processes on pp60c-src expression was assessed by comparative analysis of various adult and fetal human tissues. The maximal difference detected was a 2- to 3-fold-enhanced activity in fetal muscle compared with adult muscle. Organ-specific variations in the enzyme level were observed. Highest activity was found in brain, followed by kidney, lung, muscle, and connective tissue. Since overexpression of the cellular counterparts of viral-transforming genes may play a role in carcinogenesis, pp60c-src kinase was measured in nine spontaneous human sarcomas and 21 mammary carcinomas. Compared with the respective normal tissues and human diploid fibroblasts, 4- to 20-fold-enhanced activities were observed in one-third of the sarcomas and carcinomas. The remainder showed no or insignificantly elevated activity. The enzymes from normal and malignant tissues were indistinguishable from the virus-coded enzyme with respect to specificity for divalent cations and a predominance of phosphorylation in tyrosine. Patients carrying tumors with high pp60c-src protein kinase activity did not develop kinase-reactive antibodies against pp60c-src or pp60v-src.