Dendritic cells (DCs) loaded with tumor antigens have the potential to become a powerful tool for clinical cancer treatment. Recently, the authors showed that a tumor-specific immune response can be elicited in culture via stimulation with autologous renal tumor lysate (Tuly)-loaded DCs that were generated from cytokine-cultured adherent peripheral blood mononuclear cells (PBMCs). Here, the authors show that immunomodulatory DCs can be generated directly from nonfractionated bulk PBMC cultures. Kinetic studies of DC differentiation and maturation in PBMC cultures were performed by monitoring the acquisition of DC-associated molecules using fluorescence-activated cell sorting analysis to determine the percentage of positive immunostained cells and the mean relative linear fluorescence intensity (MRLFI). Compared with conventional adherent CD14+ cultures, which have mostly natural killer, T, and B cells removed before cytokine culture, bulk PBMC cultures exhibited an early loss of CD14+ cells (day 0 = 78.8%, day 2 = 29.6% versus day 0 = 74%, day 2 = 75%) with an increase in yield of mature DCs (DC19- CD83+) (day 5 = 17%, day 6 = 21%, day 7 = 22% versus day 5 = 11%, day 6 = 15%, day 7 = 23%). Although a comparable percentage of DCs expressing CD86+ (B7-2), CD40+, and HLA-DR+ were detected in both cultures, higher expression levels were detected in DCs derived from bulk culture (CD86 = MRLFI 3665.1 versus 2662.1 on day 6; CD40 = MRLFI 1786 versus 681.2 on day 6; HLA-DR = MRLFI 6018.2 versus 3444.9 on day 2). Cytokines involved in DC maturation were determined by polymerase chain reaction demonstrating interleukin-6 (IL-6), IL-12, interferon-gamma, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha mRNA expression by bulk culture cells during the entire 9-day culture period. This same cytokine mRNA profile was not found in the conventional adherent DC culture. Autologous renal Tuly (30 micrograms protein/10(7) PBMCs) enhanced human leukocyte antigen expression by DCs (class I = 7367.6 versus 4085.4 MRFLI; class II = 8277.2 versus 6175.7 MRFLI) and upregulated cytokine mRNAs levels. Concurrently, CD3+ CD56-, CD3+ CD25+, and CD3+ TCR+ cell populations increased and cytotoxicity against autologous renal cell carcinoma tumor target was induced. Specific cytotoxicity was augmented when cultures were boosted continuously with IL-2 (20 U/mL biological response modifier program) plus Tuly stimulation. These results suggest that nonadherent PBMCs may participate in enhancing DC maturation. Besides the simplicity of this culture technique, bulk DC cultures potentially may be used with the same efficiency as conventional purified DCs. Furthermore, bulk culture-derived DCs may be used directly in vivo as a tumor vaccine, or for further ex vivo expansion of co-cultured cytotoxic T cells to be used for adoptive immunotherapy.