Nucleic acid probes (Gen-Probe, San Diego, Calif.) can be used to identify mycobacteria in BACTEC 12B broth cultures prior to detection of growth on solid media. We developed an algorithm that can be used to make an initial choice of a probe (either Mycobacterium tuberculosis complex [MTB] or M. avium complex [MAC]) for use in testing respiratory specimens. The algorithm was based on both the fluorochrome smear result of the concentrated specimen and the time from inoculation until the BACTEC 12B broth culture is flagged (growth index 10) as presumptively positive. The MTB probe is used first for all 4+ smear specimens, 3+ smear specimens positive in 5 days, 2+ and 1+ smear specimens positive in 7 days, and smear-negative specimens positive in 11 days. The MAC probe is used for all other specimens. The algorithm is used when other information about the culture (e.g., previous positive cultures and colonial morphology of growth on solid media) is unknown. Use of the algorithm to probe 102 respiratory BACTEC 12B broth cultures (35 with MTB; 1 with MTB, MAC, and M. gordonae; 47 with MAC; and 19 with other mycobacterial species) from 1 September through 30 November 1992 resulted in the initial use of the MTB probe for 35 (97%) of the cultures positive for MTB and the use of the MAC probe for 35 (73%) of the cultures positive for MAC. Use of the algorithm aided in the efficient use of laboratory resources without delaying the time to identification of MTB isolates.