Nyk/Mer receptor tyrosine kinase is a new member of the Ufo/Axl tyrosine kinase family and is characterized by its neural cell adhesion molecule-like extracellular domain. By using a vaccinia virus expression system to express a constitutively activated form of Nyk, we identified the major sites of Nyk autophosphorylation in tryptic peptide IY749SGDY753Y754R. Tyr-749, Tyr-753, and Tyr-754 in this peptide lie in the activation loop of the kinase domain. We also studied a series of Nyk mutants in which the three tyrosine residues were replaced individually, in pairs, or all together by phenylalanine. Single mutations of Tyr-749 or Tyr-753 to phenylalanine reduced Nyk kinase activity toward exogenous substrate to 39 or 10% of that of the wild type Nyk, respectively, whereas the Tyr-754 mutant is completely inactive. All of the double and triple Tyr-Phe mutants reduced Nyk kinase activity to a level below the background. Similar results were obtained when Nyk autophosphorylation levels were examined. Our studies suggest that full activity of Nyk/Mer kinase requires phosphorylation of all three tyrosine residues in the kinase domain (Tyr-749, Tyr-753, and Tyr-754) and that Nyk kinase activity is modulated by the level of autophosphorylation in the kinase domain. Given the highly conserved nature of this region among the Ufo/Axl receptor family members, the information presented in this report may provide insight to the biochemical properties of other members of this family.