The nonequilibrium thermal dissociation (NTD) methodology has been proposed to provide a superior discrimination between specific and nonspecific hybridizations than the commonly used array techniques involving hybridization followed by a single stringent wash. Multiple studies have used this method on gel-pad, planar, and nylon membrane arrays to identify specific microbial targets in complex target mixtures. A recent physicochemical study revealed several problems, particularly when the method was used to examine complex target samples. In the present study, we investigated the effect of target concentration on NTD of complex target samples obtained from an anaerobic bioreactor. Our purpose was to experimentally demonstrate that variation in the concentrations of both specific and nonspecific targets determines the course of dissociation, which was not evaluated in initial microbiological studies. We also present an approach for analyzing the dissociation curves that is less error prone compared to those used in the previous studies. Our results show that: (i) a specific target in a mixture, at a certain concentration, may have a higher dissociation temperature/time than that of the same pure target, and (ii) the concentration dependence of the dissociation precludes usage of reference curves for identifying a target. Contrary to the previous studies, an explicit calibration is required, which makes the NTD approach impractical for high throughput analysis. © 2008 Elsevier B.V. All rights reserved.