In order to elucidate whether erythropoietin (EPO) influences male reproduction, EPO activation via erythropoietin receptor (EPOR) of Stat5 was studied in Leydig cell cultures. RT-PCR analysis of mRNA that had been prepared from primary rat Leydig cell culture and the mouse Leydig tumor cell line (I-10 cells) each demonstrated amplification of the appropriate fragment for the respective genes. Both I-10 cells and primary rat Leydig cell cultures were treated with 1U/ml recombinant human erythropoietin (rHuEPO) for various time periods upto 60 min, and cytoplasmic proteins, total cellular proteins, and nuclear extracts were isolated. Expression of Stat5b protein and phosphorylated Stat5b protein were investigated by Western blotting using anti-Stat5b pAb and anti-phospho-Stat5a/b pAb. Stat5b protein exists at the cytoplasmic level irrespective of exposure to rHuEPO, while phosphorylated Stat5b protein increases upon rHuEPO stimulation. DNA binding of nuclear protein to synthetic double-stranded oligonucleotides containing a DNA response element derived from PRL-inducible element (PIE) was examined using a gel mobility shift assay. The oligonucleotide representing the PIE of the rat beta-casein promoter was demonstrated to bind to the nuclear extract of rHuEPO-treated cells, time dependently forming a single mobility shift complex X. These data suggest that EPO binds to a receptor expressed on the surface of Leydig cells. This EPOR regulates expression of specific gene, through a Jak-Stat pathway to influence male reproductive function.