Dendritic cells (DC) are professional antigen-presenting cells that are widely used in the experimental immunotherapy of cancer. For clinical use GMP-like protocols for the preparation of functionally active dendritic cells (DC) in large numbers and at high purity are needed. However, the currently available protocols have certain disadvantages. In this study we tested the generation and clinical applicability of DC from monocyte preparations produced by immunomagnetic CD14(+) selection using a semiautomated clinical scale immunomagnetic column. Peripheral blood mononuclear cells (PBMC) of 10 patients with metastatic solid tumors were used. With the immunomagnetic separation, we obtained a cell suspension of high CD14(+) purity (median 97.4%, range 94.9-99.0) with a high monocyte yield (median 82.3%, range 63.9-100.0). Differentiation of CD14(+) cells into mature monocyte-derived DC was induced by incubation with IL-4, GM-CSF, TNF-alpha, PGE(2), IL-1 beta, and IL-6. Mature DC showed a high expression of CD83, HLA-DR, and the co-stimulatory molecules CD80 and CD86. Overall CD83(+) yield was 12.1% (range 4.0-29.4). Allogeneic T stimulatory capacity could be demonstrated for all DC preparations in proliferation assays. No significant differences in marker expression or T cell stimulation was detected between fresh DC and those derived from cryopreserved immature DC. Clinical administration of autologous DC by three different parenteral routes was tolerated by all 10 patients without systemic signs of toxicity. Our results indicate that immunomagnetic isolation of CD14(+) monocytes using the CliniMACS device is a suitable method for clinical-scale generation of functional DC under GMP-grade conditions. The selection can be performed in a closed system. Therefore, immunomagnetic CD14(+) selection can be seen as an alternative way to generate DC for clinical tumor vaccination protocols.