Receptor tyrosine kinases have become important therapeutic targets because of their involvement in diseases, including cancer. Kinase domains, which are soluble and easily purified, have found widespread use in enzyme inhibitor assays, but these domains do not exhibit full function because they are isolated from the membrane. To address this shortcoming, the authors developed a simple method to restore biologically relevant function by assembling kinase domains on a nanometer-scale template, which imitates the membrane surface. Autophosphorylation of template-assembled tyrosine kinase domains from the insulin, EphB2, and Tie2 receptors led to substantially larger phosphorylation levels compared with domains assayed under conventional conditions. Template-directed assembly increased the total substrate phosphorylation of the insulin and EphB2 receptor kinase domains as much as 60-fold and 15-fold, respectively. In contrast, substrate phosphorylation by template-assembled Tie2 was much lower than conventional conditions. The lower activity observed with the template is more biologically relevant because autophosphorylation of Tie2 is self-inhibitory. These results, as well as the underlying similarity between the organization of template-assembled and natural membrane signaling environments, suggest that template-directed assembly of signaling proteins will provide widespread benefit to basic and applied signal transduction research, especially drug discovery.