The glycolytic enzyme phosphoglycerate kinase (PGK) consists of two isozymes, somatic-type PGK-1 and testis-specific PGK-2. The isozyme switch from PGK-1 to PGK-2 occurs during spermatogenesis at the mRNA level. The distal upstream region of the gene encoding mouse PGK-2 (Pgk-2) possesses a silencer-like negative cis element. In the present study, a positive cis element located in the proximal upstream region and factor(s) bound to it were analyzed in vitro. Cell-free transcription using nuclear extracts of rat organs demonstrated that the region between nucleotide positions -82 and -64, relative to the most distal transcription initiation site at +1, stimulates transcription in testis extracts. The cis element did not act on the promoter of the thymidine kinase gene, suggesting that it stimulates Pgk-2 transcription in a promoter-specific manner. The cis element bound a nuclear factor(s), which we designated TAP-1. Introducing various base substitutions within the cis element revealed that TAP-1-binding to the element requires the sequence 5'-GGAA-3', which is the binding motif for Ets oncoproteins. We previously reported that TIN-1, a transcription inhibitor of Pgk-1, binds to a sequence similar to the Ets-binding site. The addition of an oligo DNA containing the TIN-1-binding sequence of Pgk-1 prevented TAP-1 from binding to the Pgk-2 cis element, and vice versa. These results suggest that both TIN-1 and TAP-1, which are presumably involved in transcription regulation of the two Pgk genes, recognize DNA sequences related to the Ets-binding motif.