To compare the substrate specificities of the insulin and insulin-like growth factor 1 (IGF-1) receptor tyrosine kinases, the catalytic domains of the enzymes have been expressed in Escherichia coli as fusion proteins. The purified proteins have kinase activity, demonstrating that the catalytic domain of IGF-1 receptor, like that of insulin receptor, is active independent of its ligand-binding and transmembrane domains. The specificities of the two enzymes for the divalent cations Mg2+ and Mn2+ are indistinguishable. A series of peptides has been prepared that reproduces the major phosphorylation sites of insulin receptor substrate-1, a common substrate for the two receptor tyrosine kinases in vivo. Insulin and IGF-1 receptors show distinct preferences for these peptides; whereas insulin receptor prefers peptides based on Tyr-987 or Tyr-727 of insulin receptor substrate-1, the IGF-1 receptor preferentially recognizes the Tyr-895 site. The latter site, when phosphorylated, is a binding site for the SH2 domain-containing adapter protein Grb2. The ability of the two receptor tyrosine kinases to be phosphorylated and activated by v-Src has also been examined. The catalytic activity of IGF-1 receptor is stimulated approximately 3.4-fold by treatment with purified v-Src, while insulin receptor shows very little effect of Src phosphorylation under these conditions. This observation is relevant to recent findings of IGF-1 receptor activation in Src-transformed cells, and may represent one method by which Src amplifies its mitogenic signal. Collectively the data suggest that the catalytic domains of the two receptor kinases possess inherently different substrate specificities and signaling potentials.