The folate inhibitor trimethoprim (TMP) is active against and potentially cidal to a few higher microorganisms and a wide spectrum of pathogenic bacteria except for Bacteroides, Branhamella, Brucella, Chlamydia, Clostridium, Mycobacterium, Mycoplasma, Nocardia, Neisseria, Pseudomonas and Treponema. These organisms tend to be more sensitive to sulfonamides (SUL) than to TMP, whereas TMP is 10- to 100-fold more active than SUL against most other bacteria. Synergy between TMP and SUL occurs at drug concentrations equal to or less than their respective MICs and is often seen in vitro with isolates that are sensitive or moderately resistant to one or both of the components. Synergy occurs over a wide range of ratios between TMP and SUL, the optimal being that between their respective MICs when acting singly. In vitro synergy is more impaired by bacterial resistance than by suboptimal TMP:SUL ratios. The vast majority of clinical isolates of Haemophilus, staphylococci, streptococci and enteric bacteria are inhibited in vitro by the minimum concentrations of drug attained in plasma during therapy. Exceptions are found among Enterobacter, Citrobacter, Serratia, Proteus and in particular Klebsiella where SUL resistance is common and isolates with TMP MICs of 5 mg/l or more may occur and lead to failure of TMP-SUL therapy in systemic infections. In the urinary tract drug concentrations that are synergistic and therefore inhibitory in vitro against isolates moderately resistant to SUL (MIC less than or equal to 1 g/l) and/or TMP (MIC less than or equal to 0.1 g/l) are present during TMP-SUL therapy. However, whether the synergy and the bactericidal effect of TMP-SUL observed in vitro play a role in vivo is controversial.