Dendritic cells (DC) are antigen‐presenting cells characteristic in their rarity and potent ability to stimulate resting T cells. We established several cell lines from purified mouse spleen DC and Langerhans' cells (LC) by infecting them with recombinant SV40ori⁻ adenovirus. Using this method, cells from these fractions are efficiently transformed at a frequency as high as 5 × 10⁻⁶. Some established lines have dendritic morphology, express surface markers specific for DC (defined by mAb 33D1 and NLDC145) and weak substrate adherence. Interestingly, these lines simultaneously express B cell markers (B220 and Ly‐1) and/or macrophage markers (Mac‐l and F4/80). Among the lines, OT6.2 and OT8.3.1 have retained stimulating activity in mixed lymphocyte reactions (MLR) which is attributable to the primary T cell stimulating activity of native DC However, these lines also present soluble antigens to T cell clones, a secondary T cell stimulating activity, characteristic of B cells and macrophages. Although transformation has perturbed the functional discreteness of the DC cell lineage. DC‐specific properties are partially preserved in the cell lines. These cell lines may provide useful experimental systems for the elucidation of molecular mechanisms involved in the MLR and the differentiation of DC.