By transforming two methionine auxotrophic mutants from fission yeast Schizosaccharomyces pombe with a wild-type gene library, we defined two genes, met9 and met11, which both encode a methylenetetrahydrofolate reductase. The genes cannot complement each other. We detected single transcripts for both. In vitro measurements of enzymatic activities showed that the met11-encoded enzyme was responsible for only 15-20% of the total methylenetetrahydrofolate reductase activity. A strain in which gene met9 was disrupted required significantly more methionine for full growth and efficient mating and sporulation than the strain disrupted for gene met11. The in vitro and in vivo data thus indicated that met9 was the major expressed gene. Our results are in accordance with the assumption that the two methylenetetrahydrofolate reductases generate the methyl groups necessary for methionine synthetase to convert homocysteine to methionine, and suggest that expression of the two genes is an important parameter in the control of methionine biosynthesis.