METHODS The resurgence of tuberculosis and the increase of Mycobacterium avium complex infections have renewed interest in developing more efficient systems for isolation of mycobacteria. The Bactec radiometric method enhances the recovery and shortens the detection time of mycobacteria; however, many clinical laboratories are subject to severe restrictions on the utilization of 14C radiolabelled culture media. OBJECTIVE To compare the biphasic MB-Check system in the recovery of mycobacteria from clinical specimens with the Bactec-460 system and Lowenstein-Jensen (LJ) medium. METHODS The MB-Check was evaluated for routine use in a clinical laboratory by comparing the rates and times for isolation of mycobacteria from 1840 clinical specimens with results obtained using the other systems. The isolated organisms were identified by standard procedures and specific deoxyribonucleic acid (DNA) probes. RESULTS The rate of recovery of all mycobacteria with the MB-Check system was 84.8%, compared to 87.5% for the Bactec-460 system and only 64.2% on LJ. Of the 147 M. tuberculosis isolates recovered by all methods combined, 92.5% were recovered by MB-Check, 87.1% by Bactec and 79.6% on LJ. Of the 109 isolates of nontuberculous mycobacteria (NTM) recovered by all methods combined, 44% were recovered on LJ, 75% by MB-Check and 88% by Bactec (P < 0.001). A combination of the Bactec and MB-Check systems allowed recovery of 99.2% of isolates. The Bactec system showed a faster detection time than the other two methods. CONCLUSIONS The MB-Check and Bactec systems were more efficient than LJ medium for the isolation of both M. tuberculosis and NTM. The performance of the MB-Check system and the ease with which processing, inspection, detection, and isolation can be performed, indicated that the biphasic approach for cultivation of mycobacteria is feasible and practical. The MB-Check system is more suitable than Bactec system for laboratories that have a small number of specimens or in situations where the disposal of radioactive waste is a problem.