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We evaluated the efficacy of different neutralizing solutions for use in disinfectant efficacy assays. Our approach used comparisons between the recoveries of low inocula in different treatment populations. The challenge organisms employed were those described by the AOAC for use in determining germicidal, tuberculocidal, sporicidal, and fungicidal activity of disinfectants. Neutralizer efficacy (NE) ratios were determined by comparing the recovery of identical inocula from the neutralizing solution in the presence, or the absence, of a 1:10 dilution of the biocide. Neutralizer toxicity (NT) ratios were determined between recovery of viable microorganisms incubated for a short period in peptone, and in the neutralizing medium without the biocide. An effective and non-toxic neutralizer was initially identified by NE and NT ratios of > or = 0.75. Statistical evaluation of the data was performed by ANOVA, with Dunnett's test for multiple comparisons used to confirm failures. By this analysis, 239/244 identified failures were confirmed by ANOVA of 588 NT and NE comparisons (5 presumptive failures were not confirmed by statistical analysis). We therefore conclude that recovery of 75% is a suitable criterion (2% false negative rate) for neutralizer evaluations. There was a wide degree of variability seen among the responses of different test organisms to the different recovery broths. Pseudomonas aeruginosa and Salmonella choleraesuis were particularly sensitive to commercial neutralizer broths. Interestingly, no commercially available neutralizer proved adequate for all index organisms against all biocides tested. This finding underscores the need to evaluate potential neutralizers against all microorganisms and biocides employed.