To increase the sensitivity and throughput of protein tyrosine kinase (PTK), simple, homogeneous, nonradioactive, direct and indirect fluorescence polarization (FP) protein tyrosine kinase immunoassays have been developed that are compatible with high-throughput and ultrahigh-throughput screening for developing drugs. In the direct method, a fluorescinylated peptide substrate is incubated with the kinase, ATP, and antiphosphotyrosine antibody. The phosphorylated peptide product is immunocomplexed with the antiphosphotyrosine antibody, resulting in an increase in the polarization signal. Since the direct method can be used only with a peptide substrate and requires large amounts of antiphosphotyrosine antibody, a modified indirect method, wherein a phosphorylated peptide or protein produced by kinase reaction will compete with a fluorescent phosphopeptide used as a tracer for immunocomplex formation with phosphotyrosine antibody, was developed. In this format kinase activity will result in loss of the polarization signal. Both the direct and indirect FP-PTK immunoassays have been compared with a more commonly used (32)PO(4) transfer assay and validated using lymphoid T-cell protein tyrosine kinase (Lck). In both assays, Lck activity showed a similar dependence on ATP, Lck enzyme, and peptide substrate concentration, comparable to the (32)PO(4) transfer assay. Inhibition by staurosporine and the Lck inhibitor 4-amino-5-(methylphenyl)-7-(tert-butyl)pyrazolo[3, 4-d]pyrimidine in these two FP assays was similar to that obtained in the (32)PO(4) transfer assay. The advantages of these FP-PTK assays over the other kinase assays, besides high sensitivity, are use of inexpensive nonisotropic substrate; environmental safety; homogeneous nature of FP kinase assays that are done in the same tube (or in a well of 96- or 384-well microtiter plates), without separation, precipitation, or washing; and increase of throughput.