Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25), a member of the DEAD-box protein family, is essential for completion of spermatogenesis. GRTH is present in the cytoplasm and nucleus of meiotic spermatocytes and round spermatids and functions as a component of mRNP particles, implicating its post-transcriptional regulatory roles in germ cells. In this study, GRTH antibodies specific to N- or C-terminal sequences showed differential subcellular expression of GRTH 56- and 61-kDa species in nucleus and cytoplasm, respectively, of rodent testis and transfected COS1 cells. The 56-kDa nuclear species interacted with CRM1 and participated in mRNA transport. The phosphorylated cytoplasmic 61-kDa species was associated with polyribosomes. Confocal studies on COS-1 cells showed that GRTH-GFP was retained in the nucleus by treatment with a RNA polymerase inhibitor or the nuclear protein export inhibitor. This indicated that GRTH is a shuttling protein associated with RNA export. The N-terminal leucine-rich region (61-74 amino acids) was identified as the nuclear export signal that participated in CRM1-dependent nuclear export pathway. Deletion analysis identified a 14-amino acid GRTH sequence (100-114 amino acids) as a nuclear localization signal. GRTH selectively regulated the translation of specific genes including histone 4 and HMG2 in germ cells. In addition, GRTH participated in the nuclear export of RNA messages (PGK2, tACE, and TP2) in a gene-specific manner. These studies strongly indicate that the mammalian GRTH/Ddx25 gene is a multifunctional RNA helicase that is an essential regulator of sperm maturation.