N-Myristoylation of p56lck, a member of the Src family of protein-tyrosine kinases, is essential for its proper targeting to the plasma membrane. 2-Hydroxymyristic acid (HMA) is an analog of myristic acid that becomes metabolically activated in cells to form 2-hydroxymyristoyl-CoA, a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that catalyzes protein N-myristoylation [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1990) Biochemistry 29, 10566]. In the presence of HMA, LSTRA cells, which overexpress p56lck, synthesized nonmyristoylated p56lck, which displayed a reduced electrophoretic mobility on SDS-polyacrylamide gels identical to that of a nonmyristoylated Gly2-->Ala2 mutant of p56lck. Treatment with myristic acid, 2-hydroxypalmitic acid, or 2-fluoromyristic acid did not result in the synthesis of nonmyristoylated p56lck. In contrast to the membrane-associated, myristoylated p56lck, nonmyristoylated p56lck was cytosolic. Although nonmyristoylated p56lck retained tyrosine kinase activity, it was not labeled in vivo with [32P]orthophosphate, indicating that a change in subcellular location altered its state of phosphorylation. A pulse-chase analysis revealed that cytosolic, nonmyristoylated p56lck was less stable than the myristoylated enzyme. In cell lines that do not overexpress p56lck, HMA treatment resulted in a reduction in the levels of both newly synthesized and total p56lck. Treatment of CD4+ cells with HMA caused a corresponding decrease in the amount of CD4-associated p56lck. Thus, chemical inhibition of protein N-myristoylation with HMA is an effective method for reducing the amount of p56lck available at the plasma membrane for signal transduction.