The virulence of different isolates of Mycobacterium has been associated with two morphologically distinguishable colonial variants: opaque (SmOp) and transparent (SmTr). In this report we used an in vitro assay to compare macrophage (M) responses to SmOp and SmTr Mycobacterium fortuitum variants, taking advantage of the fact that these variants were derived from the same isolate. Cells preactivated or not with gamma interferon (IFN-␥) were infected with SmOp or SmTr M. fortuitum. We showed that SmOp and SmTr induced different levels of nitric oxide (NO) production by IFN-␥-stimulated M. Indeed, the amount of IFN-␥-induced NO production by J774 cells was 4.8 to 9.0 times higher by SmOp (23.1 to 37.7 M) compared to SmTr infection (3.9 to 4.8 M) (P ‫؍‬ 0.0332),