Dendritic cells, well‐known for their potent antigen‐presenting activity, are generally present at very low frequency in the spleens of naive mice. We examined the ability of mice to generate functional dendritic cells (DC) following exposure to the cytokines interleukin‐3 (IL‐3) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF). Tumours secreting these cytokines provided a continuous stimulus resulting in a greatly increased number and frequency of DC in the spleen. These cells were purified by conventional DC isolation techniques and were found to exhibit many of the characteristics of DC from unmanipulated mice, including high allo‐stimulatory activity in mixed lymphocyte reactions and expression of many similar cell surface markers. Using ovalbumin‐peptide specific class I‐ and class II‐restricted hybridomas containing the lacZ reporter gene, we found that these cytokine‐generated DC had a greatly increased efficacy in the uptake and processing of particulate antigen. These cells appear to have retained the ability to ingest antigen that is generally associated with immature DC, but also exhibit the peptide/major histocompatibility complex (MHC)‐presenting capabilities of mature DC. Development of an assay to measure the activity of a single DC revealed that these dual activities were the properties of the majority of the cytokine‐generated DC. These findings indicate that exposure in vivo to the cytokines IL‐3 and GM‐CSF can result in the generation of large numbers of DC with increased capability of stimulating T cells. Thus, these cells may be important in vivo in the process of cross‐priming and the subsequent generation of tumour‐reactive cytotoxic T lymphocytes (CTL).