To date, it has been problematic to accurately quantify multiple nucleic acid sequences, representing microbial targets, in multi-target mixtures using oligonucleotide microarrays, primarily due to nonspecific target binding (i.e., cross-hybridization). While some studies ignore the effects of nonspecific binding, other studies have developed approaches to minimize nonspecific binding, such as physical modeling to design highly specific probes, subtracting nonspecific signal using mismatch probes, and/or removing nonspecific duplexes by scanning through a range of wash stringencies. We have developed an alternative approach that, in contrast to previous approaches, uses nonspecific target binding as a source of information. Specifically, the new approach uses hybridization patterns (fingerprints) to quantify specific nucleic acid targets in complex target mixtures. We evaluated the approach by mixing together in vitro transcribed 28S rRNA targets at varying concentrations (up to 1.0 nM), and hybridizing the 24 mixtures to microarrays (n = 3160 probes, in duplicate). Three independent Latin-square-designed experiments revealed accurate quantification of the targets. The regression between actual concentration of targets and those determined by the approach were highly positively correlated with high R2 values (e.g., R2 = 0.90, n = 6 targets; R2 = 0.84, n = 8 targets; R2 = 0.82, n = 10 targets). © 2008 Elsevier B.V. All rights reserved.