The percentage of clinical isolates of several species of Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumoniae, resistant to trimethoprim (TMPR) has increased gradually at the Brigham and Women's Hospital (Boston) in recent years. Thirty-seven of 42 TMPR isolates from six species of gram-negative bacilli conjugally transferred TMP resistance to K12 E. coli. beta-Lactam resistance cotransferred from 21 of the 37 donors, and sulfamethoxazole (SMZ) resistance cotransferred from five of the 37 donors. Plasmids that encoded TMP resistance either alone or with SMZ resistance had a molecular size of approximately 52.5 kilobases, with identical restriction endonuclease-generated "fingerprints." Plasmids encoding beta-lactam-mediated resistance (beta R) were approximately four kilobases larger and had fragment patterns that were identical for all of the TMPR/beta R plasmids tested and had many restriction endonuclease-generated bands in common with TMPR plasmids. Radiolabeled dihydrofolate reductase (DHFR) probes identified the type II DHFR as the determinant of TMP resistance. In contrast with reports from Europe, TMP resistance in multiple species of Enterobacteriaceae was found to be spread in one hospital by a single, stable conjugative plasmid that has a wide host range and encodes the type II DHFR gene.