The increment of infections produced by opportunist environmental mycobacteria (OEM) raises the question about the appropriateness of the conventional culture methods since they were initially designed to isolate M. tuberculosis. In this study we have comparatively evaluated the yield of a conventional culture (Löwenstein-Jensen) with respect to that of Middlebrook 7H12 (Bactec) in 396 selected samples in which OEM were isolated. Samples with commensal flora were previously subjected to a chemical homogenization-decontamination process with alkaline N-acetyl-cysteine. All cultures were sown with standard inoculates obtained from centrifugate material. The 7H12 culture showed a more rapid detection (mean: 17-18 days) and a greater effectiveness in the isolation of eventually pathogenic mycobacteria than the conventional Löwenstein-Jensen culture. The 7H12/LJ ratio for the species with high clinical relevance was: 1.3 for M. kansasii, 1.0 for M. marinum, 1.8 for M. scrofulaceum, 1.3 for M. xenopi, 1.5 for M. avium-intracellulare, 0.5 for M. chelonae, and 2.0 for M. fortuitum.