Cooperativity in the binding of two substrates to an enzyme is a now well-established phenomenon. The x-ray crystallographic structure of the E. coli DHFR binary TMP complex compared with the ternary enzyme-NADPH-TMP complex suggests without too imaginative extrapolation, that the conformational changes resulting from the binding of one ligand aid in favorably positioning potential binding sites for the second ligand. Of greater importance is the fact that the extent to which inhibitor binding is enhanced by the binding of NADPH varies from species to species. To a significant extent, for example, the selectivity of TMP is enhanced by the increase in its binding to the E. coli enzyme when NADPH is present as compared with several mammalian enzymes. The reverse, negative cooperativity (a decrease in binding of a substance when moving from the binary to a ternary complex), is perhaps less common and certainly less well studied. The present paper deals with one such enzyme, the DHFR from C. albicans, and by reference to another, that from S. cerevisiae, where it is shown that the binding of substrates exhibit strong negative cooperativity. It was of interest also to determine the relationship between inhibitor/NADPH cooperativity and the relative insensitivity of N. gonorrhoeae to TMP. Equilibrium studies show that the binding of TMP in binary complex with this enzyme is exceedingly poor and that a 2,200-fold cooperative effect brings the gonococcal enzyme Ki within one order of magnitude of the E. coli enzyme Ki. Even so, it takes synergism of another sort (with sulfamethoxazole) and high doses to make co-trimoxazole therapy feasible for treating gonorrhoeae. The comparative results on the gonococcal enzyme for a family of near relatives of TMP are of interest also for the reason that the structure-activity relationships with this enzyme are quite different from those of the E. coli and other microbial enzymes. Finally, it should be pointed out that although the negative cooperativity found for the candida and saccharomyces enzymes is relatively large, it is the values of the substrate Michaelis constants that are physiologically relevant. The Km values of the yeast enzymes are within the range for other DHFR and therefore the intracellular activity of the enzymes should not be compromised.