Dendritic cells (DC) are potent antigen-presenting cells with the unique capacity to initiate primary immune responses. As a result, DC are currently used in clinical studies to induce immunity against infectious disease and malignant cells. However, multiple DC subsets exist and it has been suggested that the type of DC may affect the immune response induced. The vast majority of DC used in experimental mouse tumor models is derived from bone marrow progenitors. In contrast, most in vitro as well as in vivo human studies involve the use of DC generated from adherent peripheral blood-derived monocytes in the presence of GM-CSF and IL-4. In the current report, we describe for the first time the generation and characterization of mouse monocyte-derived DC (MODC). The results indicate that mouse MODC display similar morphology, phenotype and immunostimulatory activity as compared to bone marrow-derived DC. Both DC subsets were able to efficiently take up and subsequently cross-present protein antigen to cytotoxic T cells. Moreover, we demonstrate that vaccination with peptide-loaded MODC mediates induction of tumor-reactive immunity in vivo. The isolation and characterization of mouse MODC will provide a valuable research tool to investigate fundamental aspects of DC biology and which DC subsets are most suitable to induce anti-tumor immunity.