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It is established that duplication in vitro of that amount of Ca21 (2.5 mM) and Mg2e (1.5 mM) present in blood permits vegetative growth of Yersinia pestis with repression of virulence factors encoded by the Lcr plasmid (Lcr+); similar simulation of intracellular fluid (no Ca2+ and 20 mM Mg2+) promotes bacteriostasis with induction of these virulence determinants. However, proliferation of yersiniae in mice occurs primarily within necrotic focal lesions (supplied by Ca2+-deficient host cell cytoplasm) within visceral organs rather than in Ca2+-sufficient blood. The present study addressed this enigma by defining conditions necessary for achieving vegetative growth of Lcr+ yersiniae at 37°C in simulated intracellular fluid. Maximum optical densities were increased by substitution of K+ for Na+ and elimination of Cl-; the combination of Na+ plus L-glutamate was selectively toxic to Lcr+ cells. This phenomenon was attributed in part to the absence of aspartase in Y. pestis (a lesion known to facilitate massive accumulation of L-aspartate via transamination of the oxalacetate pool by L-glutamate). Replacement of L-glutamate by exogenous L-aspartate or a-ketoglutarate reversed this toxicity by favoring retention of oxalacetate. Proliferation of Lcr+ cells in a medium containing K+ and L-aspartate but lacking added Ca2' and Na+ was markedly enhanced by increasing the concentration of fermentable carbohydrate. Accordingly, in the worst-case scenario (i.e., added Na+, Cl-, and L-glutamate), Lcr+ yersiniae underwent restriction of growth after one doubling, and in the best-case scenario (i.e., added K+ and L-aspartate), the organisms completed more than five doublings, thereby achieving full-scale growth. Both of these Ca2+-deficient media promoted maximum induction of Mg2+-induced V antigen, a virulence factor encoded by the Lcr plasmid.