Uncovering the secretome of Yersinia pestis is a necessary measure to better understand the virulence of this plague-causing bacterium. Using bioinformatics methods, the components of all the secretion systems known to date in the Y. pestis KIM genome were mapped, including several systems identified by this study. It was found that this organism possesses Sec, twin-arginine translocation, signal recognition particle, Omp85/YaeT, type I, type II, type III, type VI, chaperone/usher, autotransporter, and two-partner secretion (TPS) systems, but lacks a type IV secretion system. The TPS systems caught the authors' attention, for they are used by several bacterial pathogens for the secretion of large virulence determinants. Reverse transcriptase-PCR analysis confirmed the gene expression of three TPS systems in Y. pestis KIM; bioinformatics studies indicated that the exoproteins of these TPS systems are putative hemolysins and adhesins. Results thus suggest that these are functional systems, capable of secreting virulence proteins that might contribute to plague infection. This is the first report on the complete secretome of pathogenic Y. pestis KIM.