We introduced a promoter trap vector carrying a neo gene as a selectable marker into F9 cells and established several cell lines in which the expression of neo gene is under the control of an endogenous host gene that is active only in the undifferentiated F9 cells. Using one of these cell lines, G19, we isolated the integrated neo construct and its flanking host sequences by the plasmid rescue method, identified the host gene which contributes to the expression of neo gene, and named it the Zfp-57 gene. Two different Zfp-57 transcripts (1.8 and 3.2 kilobases) were identified in the undifferentiated F9 cells, and the levels of these transcripts were decreased significantly within a short time after induction of differentiation. We examined mouse organs for the presence of the Zfp-57 RNAs and found that the 1.8-kilobase RNA was detected only in the testis. The Zfp-57 cDNAs corresponding to the two different RNAs were isolated, and a comparison of the nucleotide sequences revealed that their coding regions were completely identical, but they differed both in length and in sequence of the 3'-untranslated region. The Zfp-57 cDNA encoded a protein consisting of 421 amino acids with an extremely high content of basic amino acid residues and multiple zinc finger motifs. Immunocytochemical analysis revealed that this protein is localized in the nucleus. These findings suggest that the Zfp-57 protein is a DNA-binding protein.