We sought to experimentally verify if testis specific serine/threonine kinases (Tssks) play a role in spermatogenesis and/or the regulation of sperm function. Purified Tssk proteins were obtained based on cloning and expression of mouse Tssk1 and Tssk2. Tssk1 and Tssk2 were detected in mature mouse and human sperm by western blotting. Immunofluorescence indicated that Tssk1 is distributed in the acrosome and the entire flagellum of mouse sperm while Tssk2 was mainly distributed in post-acrosomal region. There was no alteration in the distribution pattern of Tssk1 and Tssk2 in non-capacitated and capacitated sperm. Tssk2 distribution remained unchanged after induced acrosome reaction but no signals were detected in the acrosome for Tssk1, which was present before the acrosome reaction, though signals in flagellum were undisturbed. In human sperm, Tssk1 was found in neck and flagellum while Tssk2 was found in the equatorial region. Our results suggest Tssk1 and/or Tssk2 do play an important role(s) in the regulation of sperm function.