The detection of active plaque in nature relies primarily on demonstration of the etiologic agent of the disease. Yersinia pestis, in the flea vectors and susceptible mammalian hosts. A live animal assay is currently used for identification of a Y. pestis virulence antigen that is not expressed in the flea. We have found that DNA hybridization probes specific for Y. pestis, used in very simple sample preparation schemes, allow detection of Y. pestis in three species of fleas as well as tissues of experimentally infected mice at minimum concentrations of 1 x 10(6) bacilli/ml. We detected Y. pestis in 22 of 90 (24%) experimentally infected Xenopsylla cheopis (Rothschild), 13 of 25 (52%) Thrassis bacchi (Rothschild), and 9 of 25 (36%) Diamanus montanus (Baker), but no hybridization signals were observed from fleas that had fed on normal mice. The probe technique indicated infection in 9 of 10 potentially infected liver and spleen samples and none of the 5 control samples. Our techniques permit definitive diagnosis in 48 h.