ABSTRACT.The effects of different sulphonamides, dihydrofolate reductase inhibitors and other inhibitors of folate metabolism on growth ofAcanthamoeba culbertsoniin a chemically defined medium are reported. Among the sulphonamides, sulphamethoxazole and sulphadiazine were most effective followed by sulphanilamide and sulphaguanidine. Inhibition by each sulphonamide was reversed by p‐aminobenzoic acid as well as folic acid. 7‐Methylguanosine, a pteridine synthesis‐inhibitor, did not inhibit multiplication ofA. culbertsoni.Among the dihydrofolate reductase inhibitors, pyrimethamine blocked the amoebic growth at 100 μg/ml, while trimethoprim and cycloguanil palmoate failed to cause significant inhibition of growth even at 250 μg/ml. Metoprine inhibited amoebic growth completely at 50 μg/ml. Methotrexate and a thymidylate synthetase inhibitor 5‐fluorouracil inhibited growth strongly, with IC50values (the concentration of the drug which causes 50% inhibition of the growth at 72 h) of 1.97 and 2.45 μg/ml, respectively. Inhibition by methotrexate, metoprine or 5‐fluorouracil could not be reversed by folic acid, folinic acid, thymidine, or folinic acid plus thymidine. the results indicate unusual features inA. culbertsonifola