Using cDNA microarray hybridization from a human testicular cDNA library, a novel gene exhibiting 30-fold difference in expression level between adult and embryo human testes was cloned and named NYD-SP6, which was 1858 bp in length with 87% nucleotide identity with the mouse homologue sequence. The deduced protein structure of NYD-SP6 was found to contain two plant homeodomain (PHD) finger domains, believed to be involved in activating transcriptional regulation. Tissue distribution analysis using Northern blot indicated that the NYD-SP6 gene was expressed in a wide range of tissues, with a high expression level in the testis. Its expression in human and mouse testes by in situ hybridization was confined to Sertoli cells and the expression was developmentally regulated as demonstrated by cDNA microarray, in situ hybridization, and semiquantitative PCR in mouse testes. GFP/NYD-SP6 protein was predominantly localized in the nucleus of the transfected CHO cells, indicating its role in transcriptional regulation. In contrast, the N-terminal truncated NYD-SP6 (tNYD-SP6) localized in the nuclear envelope, indicating this region function as a nuclear localization signal. Further Northern blot analysis of gene expression in patients with spermatogenesis arrest revealed that NYD-SP6 was absent in one patient whose spermatogenesis was blocked at the stage of spermatocytes. Taken together, these results suggest that the putative protein of NYD-SP6 may play an important role in stimulating transcription involved in testicular development and/or spermatogenesis.