Immunosuppressive macrophages (Mφs) generated in the spleen of Mycobacterium intracellulare (Min)-infected mice (Min-Mφs) exhibit suppressor activity against T cell mitogenesis in response to concanavalin A. Here, we attempted to fractionate the Min-Mφs population generated in Min-infected BALB/c mice at week 2 after infection. Min-Mφs were found to consist of two distinct subpopulations differing in their adhesiveness to plastic wells. The strongly adhesive Mφs population (SA-Mφs) exhibited marked suppressor activity. The weakly adhesive Mφ population (WA-Mφs) exhibited much less suppressor activity. Both SA-Mφs and WA-Mφs possessed a CDllb+F4/80+SR-AI+CDl4+ CD206+ phenotype characteristic of matured Mφs, generated reactive oxygen intermediates, and suppressed IL-2 receptor expression by concanavalin A-stimulated T cells. Moreover, their suppressor activities were dependent on prostaglandin. These findings indicate that Min infection in mice generates two types of immuosuppressive Mφs with different levels of suppressor activity.