A medium for the selective isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum (MAIS) was developed, based upon the ability of these mycobacteria to utilize Tween 80 as sole carbon source and grow optimally at pH 5.5 on a simple mineral salts medium. Representative MAIS strains had higher efficiencies of plating on the Tween 80 medium compared with Middlebrook 7H10. It was shown that nonmycobacterial organisms in natural waters had lower efficiencies of plating on the Tween 80 medium and smaller colonies, thus allowing direct isolation and enumeration of the slowly growing mycobacteria without overgrowth.