Chemokines are key regulators of hematopoiesis and host defense. We report here that a functional expression of the chemokine receptor CXCR4 on human immature CD34(+) hematopoietic progenitors was increased as a result of sustained elevation in cellular cAMP by dbcAMP and prostaglandin E2. This effect of cAMP was specifically mediated by PKCzeta activity. CXCR4 expression and PKCzeta activation by cAMP were decreased following inhibition of cAMP effector-Rap1 by Spa1 overexpression. Interference with activation of Rac1, a downstream target of Rap1, prevented the cAMP-induced rise in PKCzeta activity and CXCR4 levels. Functional manifestation of the effects of cAMP-elevating agents revealed increased ability of human CD34(+) cells to trans-migrate the bone marrow (BM) endothelial layer and adhere to BM stroma in vitro, and augmented homing potential to the BM and spleen of immunodeficient mice in a Rac1- and PKCz-dependent manner. cAMP and TNFalpha stimulated pathways converged in PKCzeta-activated CXCR4 expression and MMP-2/9 secretion. cAMP treatment had a beneficial effect on CD34(+) cell survival in a PKCzeta-mediated fashion. Taken together, our data reveal major roles for cAMP-induced PKCzeta activation in signaling governing the motility and development of CD34(+) cells.