Isolation of mycobacteria on Löwenstein-Jensen media (LJ) and in the BACTEC MB9000 (MB) system was compared. Of 2,271 specimens, 317 were positive for 331 mycobacteria isolated in 1 or both media. The MB was positive in 238 isolates, and LJ was positive for 239 isolates; 92 isolates were detected by MB only and 93 by LJ only. Of the 331 isolates, 146 were recovered by both media. MB recovered 38 of 38 Mycobacterium tuberculosis complex isolates, while LJ recovered 23. MB recovered 94.1% (96/102) of Mycobacterium avium complex isolates and LJ 69.6% (71/102). The MB recovered 81% (65/80) Mycobacterium fortuitum-chelonae isolates and LJ 68% (54/80). Of the remaining species, MB isolated 39, while LJ isolated 91. Only 1 organism that was isolated on LJ alone was medically significant based on medical record review. The addition of LJ media to the MB9000 system is not warranted, as it causes clinically irrelevant workload, increased expenditures for the laboratory, and could cause the inappropriate treatment of patients.