A sequence encoding dihydrofolate reductase (DHFR) was isolated from a Schizosaccharomyces pombe cDNA library by selecting for trimethoprim resistance in Escherichia coli. The sequence was found to be functional in both Saccharomyces cerevisiae and Sz. pombe. When present on a multicopy plasmid, it confers increased resistance to concentrations of the drug methotrexate that are otherwise inhibitory for the standard yeast strains. The sequence was mapped by DNA hybridizations between genes adh1 and ade5 on chromosome III of Sz. pombe. The 1.6-kb insert contains a 1.5-kb open reading frame (ORF) with strong sequence similarity to other described DHFR-encoding genes. The similarity, however, is limited to a 678-bp sequence, occupying the 3'-half of the ORF. No similarity to other described DNA sequences or proteins could be found for the 5'-half. Southern and Northern blots indicate that the entire insert is present intact in the Sz. pombe genome and produces a 1.7-kb RNA transcript.