We previously identified testicular protein kinase 1 (TESK1), which phosphorylates cofilin and induces actin cytoskeletal reorganization. We now report identification and characterization of another member of a TESK family, testicular protein kinase 2 (TESK2), with 48% amino acid identity with TESK1. Like TESK1, TESK2 phosphorylated cofilin specifically at Ser-3 and induced formation of actin stress fibers and focal adhesions. Both TESK1 and TESK2 are highly expressed in the testis, but in contrast to TESK1, which is predominantly expressed in testicular germ cells, TESK2 is expressed predominantly in nongerminal Sertoli cells. Thus, TESK1 and TESK2 seem to play distinct roles in spermatogenesis. In HeLa cells, TESK1 was localized mainly in the cytoplasm, whereas TESK2 was localized mainly in the nucleus, which means that TESK1 and TESK2 likely have distinct cellular functions. Because the kinase-inactive mutant of TESK2 was localized in the cytoplasm, nuclear/cytoplasmic localization of TESK2 depends on its kinase activity. A TESK2 mutant lacking the C-terminal noncatalytic region had about a 10-fold higher kinase activity in vitro and, when expressed in HeLa cells, induced punctate actin aggregates in the cytoplasm and unusual condensation and fragmentation of nuclei, followed by apoptosis. Thus, we propose that the C-terminal region plays important roles in regulating the kinase activity and cellular functions of TESK2.