Select Agents are defined by CDC and the USDA Animal and Plant Health Inspection Service (APHIS) as biological agents or toxins deemed a threat to public, animal, or plant health, or to animal or plant products. They are classified on the basis of their ease of dissemination, mortality/morbidity rate, and potential for social disruption. A subset of these agents includes Bacillus anthracis, Yersinia pestis, Francisella tularensis, ricin toxin (RT), and staphylococcal enterotoxin B (SEB). Infection or intoxication with these agents has been shown to elicit an antigen-specific serum IgG response. We describe a fluorescent covalent microsphere immunoassay (FCMIA) for measurement of specific IgG antibodies to seven different antigens from five different select agents; B. anthracis [protective antigen (PA) and lethal factor (LF)], Y. pestis (F1 and V antigens), F. tularensis, RT and SEB simultaneously in human B. anthracis vaccinee sera (containing anti-PA and anti-LF IgG) which had been spiked with animal specific IgG antibodies to the other select agents. Inter-assay and intra-assay coefficients of variation were 6.5 and 13.4%, respectively (N = 4). There were no significant differences (P greater than 0.70) between assay responses when the assays were performed individually or multiplexed. When the observed versus expected interpolated concentrations were compared, highly linear relationships were observed (r2 values from 0.981 to 0.999, P less than 0.001). Minimum detectable concentrations (MDC) ranged from 0.3 ng mL(-1) (Y. pestis F1) to 300 ng mL(-1) (RT). Finally, the curves showed responses were linear for most analytes from their MDC to 125 (SEB) to 1,300 (Y. pestis F1) X their MDC. These data indicate that multiplexed FCMIA is a sensitive and accurate method for simultaneous measurement of specific IgG in serum to CDC select agents and may be of value in screening either decontamination workers or the general population for exposure to/infection with these agents.