International audience ; In this study, 2275 clinical isolates of 49 species of non-tuberculous mycobacteria isolated in The Netherlands were subjected to standardised drug susceptibility testing using the Middlebrook 7H10 agar dilution method. Clarithromycin and rifabutin were most active, with 87% and 83% of all isolates, respectively, being susceptible. Susceptibility to ciprofloxacin (44%) and amikacin (32%) was limited and was mostly restricted to , , and phylogenetically related species. Susceptibility to isoniazid (0.5%), rifampicin (37%), ethambutol (35%) and streptomycin (33%) was rare; susceptibility to cycloserine, clofazimine and prothionamide was generally restricted to slow growers, although prothionamide also had activity against and related species. Significant discrepancies between in vitro and in vivo activity exist. To improve the utility of drug susceptibility testing, the selection of drugs should be changed to more drugs with proven clinical efficacy correlating with in vitro susceptibility.