The most important mechanism of trimethoprim (Tp) resistance is the plasmid-encoded production of an additional Tp resistant dihydrofolate reductase (DHFR). The epidemiology of plasmid-mediated resistance to Tp has been studied by biochemical typing of the enzymes responsible. In recent years several new Tp resistant DHFRs have been identified. The type III enzyme, considered the rarest of the plasmid-mediated DHRFs was isolated once in New Zealand in 1979 and never subsequently detected. However the biochemical properties of a Tp resistant DHFR isolated in Nottingham were examined as DNA gene probing had suggested that the enzyme was a type III and the biochemical properties confirmed this. The enzymes responsible for Tp resistance in two outbreaks of Shigella in the United States were examined. Detailed biochemical analysis suggested that the two enzymes were different from each other but similar to the type III. Therefore the three enzymes were subsequently renamed types IIIa, IIIb and IIIc. The properties of the type IIIb enzyme were very similar to the original type IIIa; however, sequence analysis of the N-terminal of this protein showed that it was quite distinct from the type IIIa. The type IV DHFR was isolated in South India in 1984 and is the only inducible plasmid-mediated DHFR. Examination of the induction process suggested that the resistance mechanism of the enzyme was similar to chromosomal hyperproduction where resistance is achieved not because of the insensitivity of the DHFR, but because it is produced in such an amount that it 'swamps' the inhibiting Tp. Purification and sequence analysis of the type IV DHFR revealed that the enzyme was similar to the chromosomal DHFR and that it was complexed with NS1 an E.coli DNA binding protein. The biochemical properties of the type V DHFR, which was isolated in Sri Lanka in 1985, were similar to those of the type 1 enzyme, with the exception that the type V has an unusually low molecular mass when measured in Sephadex. On native polyacrylamide gel electrophoresis however the two enzymes co-migrate, this biochemical similarity suggests the two enzymes are closely related. The efficiency of plasmid-mediated resistance to Tp has compromised the use of this drug in many parts of the world, from bichemical studies it is clear that plasmid-mediated enzymes continue to evolve.