Dihydrofolate reductase (DHFR) gene from Saccharomyces cerevisiae was cloned by the phenotype of resistance to a folate analog, trimethoprim, and identified by restriction map and the nucleotide sequence analyses. The DHFR gene was fused to the structural region of E. coli lacZ gene, and the hybrid gene directed the synthesis of a fused protein with a DHFR moiety at the amino terminus and a β-galactosidase moiety at the carboxy terminus under the transcriptional control of yeast DHFR promoter. The transcriptional activity of the DHFR gene was compared to well-known yeast CYCI gene. When introduced into yeast cells, β-galactosidase level directed by the fused gene was about 21% of that directed by pLG669-Z which contains yeast CYCI gene fused to lacZ in a minimal medium, while it was about 39% in E. coli cells grown in LB medium containing ampicillin. Our results show that the yeast DHFR gene activity is lower than the yeast CYCI gene activity by about 5 folds in S. cerevisiae.