Dendritic cells (DC) are the professional APCs that initiate T cell immune responses. DC can develop from both myeloid and lymphoid progenitors. In the mouse, the CD8alpha(+) DC had been designated as "lymphoid" DC, and CD8alpha(-) DC as "myeloid" DC until recently when it was demonstrated that common myeloid progenitors can also give rise to CD8alpha(+) DC in bone marrow chimera mice. However, it is still not clear which committed myeloid lineages differentiate into CD8alpha(+) DC. Because monocytes can differentiate into DC in vivo, the simplest hypothesis is that the CD8alpha(+) DC can be derived from the monocyte/macrophage. In this study we show that cell clones, isolated from CD8alpha(+) DC lymphoma but with a monocytic phenotype (CD11c(low/-)D11b(high)CD8alpha(-)I-A(low)), can redifferentiate into CD8alpha(+) DC either when stimulated by LPS and CD40L or when they migrate into the lymphoid organs. Maturation of DC in vivo correlated with strong priming of allogeneic T cells. Moreover, the monocytes from cultured splenocytes or peritoneal exudates macrophages of wild-type mice are also capable of differentiating into CD11c(+)CD8alpha(+) DC after their migration into the draining lymph nodes. Our results suggest that monocytes can be direct precursors for CD11c(+)CD8alpha(+) DC in vivo. In addition, the monocyte clones described in this study may be valuable for studying the differentiation and function of CD8alpha(+) DC that mediate cross-presentation of Ag to CD8 T cells specific for cell-associate Ags.