Two strains of trimethoprim-resistant Shigella sonnei bearing R plasmids pBH600 and pBH700 each elaborated a dihydrofolate reductase (DHFR) and were moderately resistant to trimethoprim (minimum inhibitory concentrations, 128 and 256 micrograms/ml, respectively). Neither plasmid hybridized to probes for DHFR types I, II, or III. The trimethoprim resistance genes from the R plasmids resided on a 1600-base pair (bp) PstI fragment of pBH600 and an 1800-bp PstI fragment of pBH700. Isoelectric focusing showed distinct isoelectric points for the enzymes coded for on pBH600 (5.3) and pBH700 (5.6-5.7). Trimethoprim-resistant S. sonnei from 10 locations in nine states were examined. Isolates from 8 locations hybridized only to a pBH700-derived probe and one isolate hybridized to a pBH600-derived probe. These two trimethoprim resistance genes appear novel. The gene on plasmid pBH700 codes for an enzyme that seems widespread among S. sonnei isolates in the USA.