Natural competence of Streptococcus pneumoniae was used to locate and enrich DNA restriction fragments, biologically active for transformation of thymidine-deficient to thymidine-proficient cells. Mutations in the dihydrofolate reductase gene are accompanied by resistance to the drug trimethoprim (Tp). A 6.5-kb region of the pneumococcal chromosome encompassing the dihydrofolate reductase gene has been cloned in plasmid pLS1. Escherichia coli mutants, resistant to Tp, became fully sensitive to the drug when they harbored the recombinant plasmid. The pneumococcal dfrA mutation has been mapped within a 500-bp DNA region.