The human Eppin (hEppin) (SPINLW1; Epididymal protease inhibitor) gene was first described and sequenced in 2001, and later identified as an immunocontraceptive target for males in 2004. The expression and function of the mouse eppin (mEppin) gene was first described in 2003, and recent studies have shown that mEppin protein has a similar male contraceptive effect in mice. In this study, we designed a probe to detect mEppin mRNA in frozen sections from the testes of 60-day-old mice as well as in the GC-1, GC-2 and MLTC-1 cell lines, using a hyperbiotinylated oligonucleotide DNA probe. In addition to Sertoli cells, the expression of mEppin mRNA was localized to the spermatogonia, spermatocytes and Leydig cells. Subsequent RT-PCR and Western blot analysis confirmed the results of the in situ hybridization. Immunohistochemistry was performed on corresponding sections of mouse and human testis using an anti-c-kit antibody to detect spermatogonia and an anti-eppin antibody to detect Eppin protein. The expression of mEppin protein was detected in A1-A4 spermatogonia and the expression of hEppin was detected in type A spermatogonia as well as in the earliest preleptotene spermatocytes. The present study demonstrates that the expression of hEppin is similar to that of mEppin, and is localized to differentiated spermatogonia. Moreover, the findings support the hypothesis that mEppin protein is secreted from Sertoli cells and taken up by spermatogonia.