The ZNF268 gene was originally isolated from an early human embryo cDNA library. Several different transcripts have been isolated for the ZNF268 gene and developmental expression studies suggest that ZNF268 plays a role in the development of human fetal liver and the differentiation of blood cells. In our effort to study the functions of ZNF268 in different organs during development and in pathogenesis, we have now identified 3 novel splicing isoforms, ZNF268e, ZNF268f and ZNF268g, in human fetal tissues and human tumor derived cell lines. The 8 alternatively spliced mRNAs discovered so for are predicted to encode 3 protein isoforms. Expression analysis showed that different mRNA isoforms have different expression profiles. In particular, ZNF268c mRNA was detected only in tumor cells, and ZNF268f appeared to be tissue-specific. By Western blot analysis, all 3 ZNF268 protein isoforms, ZNF268a, ZNF268b1 and ZNF268b2, were expressed in tumor cell lines, while only two protein products, ZNF268b1 and ZNF268b2, were detected in human fetal tissues. Subcellular localization analysis showed that ZNF268a and ZNF268b2 distributed diffusely throughout the cell, while ZNF268b1 mainly localized in the cytoplasm. Moreover, using a CAT reporter system fused to the Gal4 DNA binding domain of the ZNF268 gene, the ZNF268a and b2 activated the CAT reporter gene expression, while the KRAB domain, corresponding to the ZNF268b1 repressed the reporter gene expression. Taken together, our results showed that multiple ZNF268 splicing products encode multiple ZNF268 protein isoforms with different subcellular localization, and that the ZNF268 gene may function as a transcriptional activator in the growth and differentiation of cells in development and/or pathogenesis.