From 1972 to 1984, all Enterobacteriaceae isolated from clinical specimens at St. Joseph Hospital in Paris were tested for susceptibility to trimethoprim. During this period, resistance to trimethoprim increased from 17.9% to 25.5%; the increase was due mainly to strains with high-level resistance. Genetic studies, including transferability, incompatibility grouping, determination of the molecular mass of plasmids, and hybridization with dihydrofolate reductase 1 and 11 genes, were performed with randomly selected strains, and the results were compared with those of similar studies in other countries. The most striking phenomenon in trimethoprim-resistant strains was the presence of various resistance mechanisms and of different plasmids, transposons, and genetic determinants.