We cloned a cDNA encoding a novel extracellular signal-regulated kinase (ERK) 2-binding protein, EBITEIN1, by yeast two-hybrid screening. Northern and Western blotting experiments showed that the transcript and protein were expressed in the testes. Furthermore, immunohistochemical experiments showed that EBITEIN1 existed at high levels in round spermatids, but at very low levels or not at all in other testicular cells. During spermatogenesis, EBITEIN1 was first translated after meiosis when cells became haploid, then the amount of EBITEIN1 protein gradually increased, reaching a maximum at Oakberg's stage 9. Subsequently, the level of EBITEIN1 decreased such that it was undetectable when the flagellum of the spermatozoon was generated. On a subcellular level, EBITEIN1 localized in the cytoplasm. Based on these results, we propose that EBITEIN1 is an interactor of ERK2 in the intracellular signal transduction pathway that occurs during the morphogenetic development of round spermatids to spermatozoa. The existence of this novel ERK2-interactor indicates that there could be a novel intracellular signaling pathway and/or regulatory mechanism by which ERK2 regulates intracellular events.