The mouse zinc-fingers and homeoboxes 1 (ZHX1) gene was cloned and its transcriptional regulatory mechanism analysed. The mouse ZHX1 gene spans approximately 29 kb and consists of five exons. Exons 1-3 contain the nucleotide sequence of the 5'-noncoding region of mouse ZHX1 cDNA, exon 4 contains a part of the 5'-noncoding region, an entire coding sequence, and a part of the 3'-noncoding sequence, and exon 5 contains the resulting 3'-noncoding sequence. The ZHX1 gene exists as one copy in the haploid mouse genome. Two species of ZHX1 mRNA with or without the nucleotide sequence of the third exon are produced by an alternative splicing. To investigate the regulatory elements involved in the transcription of the ZHX1 gene, transient DNA transfection experiments with ZHX1/firefly luciferase reporter genes were performed using a lipofection method. Functional analyses of a series of 5'- and 3'-deletion constructs of the reporter genes revealed that the nucleotide sequence between -59 and +50 is required for full promoter activity in mouse embryonal carcinoma F9 cells. Two positive regulatory cis-acting elements in the region were identified. These elements, designated as Box A and Box B, are located between nucleotides -47 and -42 and +22 and +27, respectively, and synergistically stimulate transcription of the mouse ZHX1 gene. Electrophoretic mobility shift assays with specific competitors and antibodies show that PEA3 and Yin and Yang 1 (YY1) bind to Box A and Box B, respectively. Thus, we conclude that PEA3 and YY1 synergistically stimulate the transcription of the ZHX1 gene.