The Yersinia pestis proteome was studied as a function of temperature and calcium by two-dimensional differential gel electrophoresis. Over 4,100 individual protein spots were detected, of which hundreds were differentially expressed. A total of 43 differentially expressed protein spots, representing 24 unique proteins, were identified by mass spectrometry. Differences in expression were observed for several virulence-associated factors, including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as several putative virulence factors and membrane-bound and metabolic proteins. Differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants.