Toxoplasma gondii grown on MRC5 fibroblasts in 96-well tissue culture plates was tested for susceptibility to five antimicrobial agents. T. gondii growth was quantitated by an enzyme-linked immunosorbent assay, which was performed directly on the fixed cultures, using a rabbit anti-T. gondii immunoglobulin G as the first antibody and a phosphatase-labeled anti-rabbit immunoglobulin G as the second antibody. Optical density values were highly correlated with the number of T. gondii organisms in the Giemsa-stained cultures (r = 0.89), and an enzyme-linked immunosorbent assay was used to assess the effect of antimicrobial agents at various concentrations. For each drug, regression models were used to quantify the relationship between optical density values and antimicrobial agent concentrations in the cultures. A significant inhibitory effect was found with pyrimethamine and sulfadoxine for concentrations greater than or equal to 0.05 and 30 micrograms/ml, respectively. With spiramycin, a progressive increase in inhibition of T. gondii was observed for increasing concentrations from 1 to 100 micrograms/ml. Ornidyl (difluoromethylornithine) and (2R,5R)-6-heptyne-2,5-diamine, which are ornithine decarboxylase inhibitors, were found to have a marked inhibitory effect for concentrations greater than or equal to 25 and 2 mM, respectively. This proposed method was sensitive and easy to perform and does not require the use of radiolabeled compounds; since it allows experimental design on replicate cultures and can be partially automated, it thus may prove useful for the systematic screening of the activity of new compounds against T. gondii.