AMY-1 has been identified by us as a c-Myc-binding protein and was found to stimulate c-Myc transcription activity. AMY-1 was also found to be associated with protein kinase A anchor protein 84/149 (S-AKAP84/AKAP149) in the mitochondria in somatic cells and sperm, suggesting that it plays a role in spermatogenesis. To determine the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human testis cDNA library, and a clone encoding a novel protein, AAT-1, was obtained. Three isoforms of AAT-1, AAT-1alpha, -beta, and -gamma, were found to be derived from an alternative splicing of the transcripts of the aat-1 gene, which was mapped at human chromosome 3q13-3q21. AAT-1 was found to be specifically expressed in the testis during the course of spermatogenesis and also to be present in the spermatid and mature sperm, as was AMY-1. AAT-1alpha was found to bind to and be colocalized in mitochondria with AMY-1 in human HeLa and mouse GC-1 cells. Furthermore, AAT-1alpha was found to bind to the N-terminal half of S-AKAP84/AKAP149 in a quaternary complex with AMY-1 and a regulatory subunit (RII) of cAMP-dependent kinase (PKA), in which AAT-1alpha was associated with RII via S-AKAP84/AKAP149, in rat testis and HeLa cells. It was then found that AAT-1alpha weakly stimulated a phosphorylation activity of PKA and also that AAT-1 itself was phosphorylated by PKA in vivo and in vitro. These results suggest that both AAT-1 and AMY-1 play roles in spermatogenesis.