Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA) were developed to simultaneously detect many of the CDC category "A" bioterrorism agents. The "Bio T" DNA assay was developed to detect: Variola major (VM), Bacillus anthracis (BA), Yersinia pestis (YP), Francisella tularensis (FT) and Varicella zoster virus (VZV). The "Bio T" RNA assay (mRT-PCR-EHA) was developed to detect: Ebola virus (Ebola), Lassa fever virus (Lassa), Rift Valley fever (RVF), Hantavirus Sin Nombre species (HSN) and dengue virus (serotypes 1-4). Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked) clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD)) of the DNA asssay for genomic DNA was 1x10(0)~1x10(2) copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1x10(-2) TCID(50)/mL. The LOD for recombinant controls ranged from 1x10(2)~1x10(3)copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1x10(4)~1x10(6) copies/mL without extraction and 1x10(5)~1x10(6) copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1x10(-4) dilution for dengue 1 and 2, 1x10(4) LD(50)/mL and 1x10(2) LD(50)/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1x10(3) copies/mL (1.5 input copies/reaction) for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The surrogate sensitivities of these two assays were 100% (95%CI 83-100) for FT, BA (pX02), YP, VM, VZV, dengue 2,3,4 and 95% (95%CI 75-100) for BA (pX01) and dengue 1 using spiked clinical specimens. The specificity of both BioT multiplex assays on spiked specimens was 100% (95% CI 99-100). Compared to other available assays (culture, serology, PCR, etc.) both the BioT DNA mPCR-EHA and BioT RNA mRT-PCR-EHA are rapid, sensitive and specific assays for detecting many category "A" Bioterrorism agents using a standard thermocycler.