Dendritic cells (DCs) are professional antigen-presenting cells with a highly immunostimulatory function and the capacity to activate naïve T cells. In recent years the rapid progress in mouse and human DC research can be mainly attributed to the generation of DCs from precursor cells in vitro, although a lack of reagents has hampered DC research in many large animal models. Here we describe the generation and characterization of ovine monocyte-derived DCs in vitro. In addition to the characteristic morphology and non-adherence of DCs, peripheral blood mononuclear cell monocytes cultured with ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) expressed CD11c and major histocompatibility complex (MHC) class II, but did not express CD14. High levels of endocytosis and an ability to stimulate antigen-specific proliferation of CD4 T lymphocytes were also demonstrated.