BACKGROUND AIMS: Plasmacytoid dendritic cells (pDC) are a dendritic cell (DC) subset specialized in the production of high amounts of interferon (IFN) type I (IFN-α, -β) in response to viruses. They can be purified from peripheral blood mononuclear cells (PBMC), usually using magnetic bead sorting. METHODS: In this study, we set up a counterflow centrifugal elutriation (CCE) procedure to enrich pDC from PBMC. We first analyzed each CCE fraction for the presence of pDC using CD123 and BDCA-2 as markers. We then purified pDC using CCE and magnetic beads and verified that their functions were not affected by this procedure. RESULTS: pDC were sorted by CCE into intermediate fractions between those containing lymphocytes and monocytes. The pDC frequency in these intermediate fractions was 3-fold that in PBMC. Using negative-magnetic bead sorting, starting with the same number of cells and beads, we obtained more than twice as many pDC from intermediate fractions as from PBMC. The phenotypes and IFN-α production capacities of sorted pDC from PBMC and from intermediate fractions were similar, both immediately after sorting and after stimulation with CpG-A oligodeoxynucleotides. In addition, we showed that intermediate fractions could be cryopreserved and that magnetic bead sorting could be performed with the same efficiency after thawing. CONCLUSIONS: Altogether, our results show that CCE can be used to enrich lymphocytes, monocytes and pDC from the same donor, without magnetic beads on their surface. Our method should be useful for the purification of these cells for experimental research and may also be adaptable for clinical use in immunotherapy.