We have developed a new micromethod to study the effect of drugs on microsporidia, using MRC5 fibroblasts infected by 105 spores of Encephalitozoon cuniculi. After 3 days of incubation with various concentrations of drugs, parasitic foci were counted in stained cultures. The inhibition of microsporidial growth exceeding 90% with albendazole (0.005 ,ug/ml), fumagillin (0.001 ,ug/ml), 5-fluorouracil (3 ,ug/ml), and sparfloxacin (30 ,ug/ml) was observed. Chloroquine, pefloxacin, azithromycin, and rifabutin were partially effective, at high concentrations. Arprinocid, metronidazole, minocycline, doxycycline, itraconazole, and difluoromethylornithine were not evaluable, since concentrations that inhibited microsporidia were also toxic for fibroblasts. Pyrimethamine, piritrexim, sulfonamides, paromomycin, roxithromycin, atovaquone, and flucytosine were ineffective. Our results confirm that albendazole and fumagillin have marked activity against E. cuniculi and show the antimicrosporidial activity of 5-fluorouracil and sparfloxacin. These data may form the basis for treatment of Encephalitozoon hellem and Septata intestinalis infections and represent an attempt to identify drugs effective against Enterocytozoon bieneusi.Microsporidia are emerging as opportunistic pathogens in immunocompromised patients, especially those with AIDS (7, 27). Although there have been major advances in curative treatment and prophylaxis of protozoan opportunistic infections, there is still no effective treatment for microsporidiosis; moreover, in vitro and in vivo experimental models for evaluating the activity of antimicrobial agents against these parasites are lacking. Enterocytozoon bieneusi, probably the most frequent microsporidian infecting AIDS patients, cannot be continuously cultured and does not infect laboratory animals. Other major species infecting AIDS patients are Encephalitozoon hellem (11), Septata intestinalis (3), and Encephalitozoon cuniculi (32, 38), which belong or are closely related to the getius Encephalitozoon. The conditions for tissue culture and maintenance of E. cuniculi in vitro are well established, and the effects of antimicrobial agents and disinfectants against this parasite have been evaluated previously (6,28,29,35). Albendazole, fumagillin, and to a lesser degree chloroquine were found to inhibit microsporidial growth, as assessed by a reduction in the number of infected cells or excreted spores. However, the relationship between the inhibitory effect and the concentration of the antimicrobial agent was not precisely determined, since only one or two concentrations of each drug were tested. The objective of our study was to develop a microculture method suitable for screening drugs and for characterizing their inhibitory effect on microsporidia. We first compared the growth kinetics of E. cuniculi in MDCK and MRC5 cells cultured in 24-and 96-well tissue culture plates and defined the optimum conditions for large-scale drug testing, in terms of feasibility and reproducibility. We then used this micrometh...