A novel protein, AAT-1, was identified as a AMY-1-binding protein and three splicing variants of AAT-1, AAT-1alpha, -beta and -gamma were identified. The function of AAT-1 is thought to be related to spermatogenesis. In this study, we further identified other splicing isoforms of AAT-1, AAT-1L, AAT-1M and AAT-1S, consisting of 767, 603 and 252 amino acids, respectively. These isoforms were found to use a promoter different from that used by AAT-1alpha, -beta and -gamma in the aat-1 gene, which contains 20 exons. Only 60 amino acids in the C-terminal portion of AAT-1 derived from exons 15-17 are common among AAT-1L, AAT-1M, AAT-1S and AAT-1alpha. While AAT-1alpha is specifically expressed in the testis, AAT-1L, AAT-1M, AAT-1S were found to be differentially expressed in human tissues. All of the isoforms of AAT-1 were found to bind to and colocalized with AMY-1 in human cells. While AAT-1L and AAT-1M were found to be localized diffusely in the cytoplasm, AAT-1S, like AAT-1alpha, was found to be localized in the mitochondria-like structure, suggesting different roles of AAT-1 isoforms in cells.