The mechanisms of resistance to trimethoprim (TMP) in the clinical isolates of Salmonella typhimurium were studied. The experimental results indicated that the frequency of TMP resistance of 50 strains of Salmonella typhimurium was 76%. Seven of the resistant strains were highly resistant to TMP and four of them contained different plasmids which could transfer in the same species and between different ones, and could be eliminated with 8% of SDS. The results of comparing the activities and characteristics of dihydrofolate reductase (DHFR) in crude extracts from seven resistant strains and those from controls suggested that overproduction of the chromosomal DHFR was the resistance mechanisms in three plasmid-free strains and the levels of DHFR activity of the strains was increased during prolonged exposure to TMP in vitro. However resistance to TMP of the plasmid-containing strains resulted from production of plasmid-mediated TMP resistant type Ia DHFR and a new type DHFR which has not been reported up to now. The present work provided the theoretical basis for clinical use of TMP and attempts to suppress development and spread of duge resistance of pathogenic bacteria.