Salmonellae are facultative intracellular bacteria capable of surviving within macrophages. Salmonella pathogenicity island 2 (SPI-2) is required for growth within macrophages and for virulence in mice. In this study, we show the involvement of SPI-2 in a signal transduction pathway that induces cytokine expression in Salmonella-infected macrophages. High levels of interleukin-10 (IL-10) mRNA were induced in macrophages by infection with wild-type salmonellae compared to a strain carrying a mutation in the spiC gene, which is encoded within SPI-2. The two strains had the same effect on the expression of proinflammatory cytokines such as IL-1 alpha, IL-6, and tumor necrosis factor alpha. IL-10 expression was dose dependently blocked by treatment of infected macrophages with the protein kinase A (PKA) inhibitor H-89, while IL-10 expression was increased by the PKA activator dibutyryl cyclic AMP. Cyclic AMP-dependent PKA activity was higher in macrophages infected with wild-type salmonellae compared to the spiC mutant, and Ser(132) phosphorylation of cyclic AMP response element-binding protein (CREB), which is an important mediator of PKA activation, correlated with the levels of PKA activity. Taken together, these results indicate that salmonellae cause an SPI-2-dependent increase in PKA activity that leads to CREB phosphorylation, resulting in up-regulation of IL-10 expression in Salmonella-infected macrophages. Suppression of IL-10 expression by an antisense oligonucleotide did not affect the growth of wild-type salmonellae within macrophages, whereas growth was dose dependently inhibited by H-89, suggesting that the PKA signaling pathway plays a significant role in intramacrophage Salmonella survival.