Radiometric MICs of clarithromycin, a new macrolide drug, were determined against five mycobactin-dependent strains of Mycobacterium paratuberculosis (including two Crohn's disease clinical isolates) and compared with those of other drugs which included rifampin, ethambutol, amikacin, ofloxacin, ciprofloxacin, and sparfloxacin. Among the drugs screened, clarithromycin was the drug for which MICs were lowest against the five strains tested. As MICs were significantly below the reported Cmax levels (about 4 micrograms/ml), the intracellular activity of clarithromycin against the type strain of M. paratuberculosis maintained in cultured macrophages was screened. Clarithromycin was able to kill the initial inoculum by more than 1 log within 7 days, and this activity was further potentiated by ethambutol. Extracellular drug combination screened by using sublethal concentrations of the drugs showed that ethambutol was able to enhance clarithromycin activity in three out of four M. paratuberculosis strains instead of only one out of four strains (or none in the case of ofloxacin) when associated with other drugs. These results suggest that clarithromycin may be fruitful to treat human disease in which M. paratuberculosis may be etiologically involved.