We compared the Mycobacteria Growth Indicator Tube (MGIT) system with the BACTEC 460 (B460) and Lowenstein Jensen (LJ) systems for the recovery of mycobacteria (acid-fast bacteria [AFB]) from 1,441 clinical specimens. Excluding 13 isolates of Mycobacterium gordonae, 178 significant AFB isolates were recovered from 113 patients. Isolates (119) of the Mycobacterium avium complex (MAC) accounted for 67% of all isolates, while isolates (30) of the Mycobacterium tuberculosis complex (MTB) accounted for 17% of isolates. The MGIT system recovered 98 (82%) MAC and 27 (90%) MTB isolates, while the B460 system recovered 101 (85%) MAC and 28 (93%) MTB isolates and the LJ system recovered 91 (76%) MAC and 25 (83%) MTB isolates. Overall, the MGIT system recovered 152 isolates of AFB (85.4% sensitivity), and the B460 and LJ systems recovered 151 (84.8% sensitivity) and 137 (76.9% sensitivity) AFB isolates, respectively. The recoveries of AFB with combinations of media were as follows: MGIT + LJ, 93.2%; B460 + LJ, 92.1%; and MGIT + B460, 96.6%. Although the sensitivity of MGIT was equivalent to that of B460, MGIT required a longer incubation (median, 11 days) than did B460 (median, 8 days) to become positive (P < 0.05).