Summary Most knowledge about dendritic cells (DCs) and regulatory T cells in humans has been gathered from circulating cells but little is known about their frequency and distribution in lymphoid organs. This report shows the frequency, phenotype and location of DCs and regulatory T cells in deceased organ donors' spleens. As determined by flow cytometry, conventional/myeloid DCs (cDCs) CD11chighHLA-DR+CD123−/low were 2·3 ± 0·9% and LIN- HLA-DR+CD11chigh 2·1 ± 0·3% of total spleen cells. Mature CD11chighHLA-DR+CD83+ were 1·5 ± 0·8% and 1·0 ± 1·6% immature CD11chighHLA-DR+CD83- cDC. There were 0·3 ± 0·3% plasmacytoid DCs (pDC) CD11c−/lowHLA-DR+CD123high and 0·3 ± 0·1% LIN-HLA-DR+CD123high. Cells expressing cDCs markers, BDCA-1 and BDCA-3, and pDCs markers BDCA-2 and BDCA-4 were observed in higher frequencies than DCs with other phenotypes evaluated. CD11c+, CD123+ and CD83+ cells were located in subcapsular zone, T cells areas and B-cell follicles. CD4+CD25high Tregs were 0·2 ± 0·2% and CD8+CD28- comprised 11·5 ± 8·1% of spleen lymphocytes. FOXP3+ cells were found in T- and B-cell areas. The improvement in cell separation, manipulation and expansion techniques, will facilitate the manipulation of donor spleen cells as a part of protocols for induction and maintenance of allograft tolerance or treatment of autoimmune diseases.