I developed a rapid radiometric assay to determine susceptibility of 2 slowly growing pathogenic mycobacteria, Mycobacterium paratuberculosis and Mycobacterium avium, in vitro to antimicrobial agents. Susceptibility of the mycobacteria to the antimicrobials was measured by inhibition of incorporation of ('3)H uracil into the mycobacterial RNA. This assay was compared with an agar dilution assay and showed nearly 80% overall agreement. The technique originally developed was not readily applicable to determining minimum inhibitory concentrations of antimicrobials;The rapid assay was modified and applied to (1) determine the susceptibility of M. paratuberculosis and M. avium to a large number of antimicrobials, and (2) determine, in vitro, the minimum inhibitory concentrations of antimicrobial agents to which the mycobacteria were found susceptible. Mycobacterium paratuberculosis was more refractory to antimicrobial agents than was M. avium;The entire assay was conducted in 8-ml minivials used for liquid scintillation counting. The homogeneous liquid scintillation counting system was used to rapidly, and safely evaluate a large number of antimicrobial agents. The assay was completed in 3.5 days. The time required for agar dilution assays is 6 to 12 weeks.