During our attempts to reevaluate and improve mycobacteriology laboratory procedures, we found that isolation of mycobacteria from undecontaminated specimens through the use of selective media was unrewarding and that specimen decontamination was an essential step. A reevaluation of the N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) decontamination procedure (G. P. Kubica, W. E. Dye, M. L. Cohn, and G. Middlebrook, Am. Rev. Respir. Dis. 87:775-779, 1963) indicated that this method could be simplified and shortened without affecting the isolation rate or growth and without increasing the contamination rate. The modified NALC-NaOH procedure effectively combines the decontamination step with the concentration step: specimens are mixed with the NALC-NaOH solution on a Vortex mixer and, without any waiting or addition of buffer or water to the digested specimens, centrifuged at 3,000 X g for 15 min, and the sediment is suspended in phosphate buffer (pH 5.3). The modified method is simpler, faster, and safer than the original procedure, and the reduced manipulation is also likely to minimize the chance for cross-contamination in sequential specimen processing. The total alkali exposure time in the modified NALC-NaOH method should be kept to a minimum; if necessary or feasible, the concentration of NaOH in the digestion solution may be reduced from the usual strength of 2% to about 1.5% to compensate for the longer alkali exposure time in the modified method to maintain the isolation rate or to improve it.