A bead-based liquid hybridization assay, Luminex 100, was used to identify four pathogenic bacteria, Bacillus anthracis, Clostridium botulinum, Francisella tularensis subsp. tularensis, and Yersinia pestis, and several close relatives. Hybridization between PCR-amplified target sequences and probe sequences located within the 23S ribosomal RNA gene rrl and the genes related to the toxicity of each bacterium was detected in single-probe or multiple-probe assays, depending on the organism. The lower limits of detection LLDs for the probes ranged from 0.1 to 10 ng. Sensitivity was improved using lambda exonuclease to digest the noncomplementary target strand. All contributors in 33 binary, ternary, and quaternary mixtures in which all components were present in a 1:1 ratio were identified with an 80% success rate. Twenty-eight binary mixtures in which the two components were combined in various ratios were further studied. All target sequences were detected, even when the minor component was overshadowed by a tenfold excess of the major component. Abstract published by arrangement with Wiley.