There is growing interest in the in vitro generation of dendritic cells (DC) from peripheral blood monocytes, but the effect of the method chosen to isolate CD14+ monocytes for subsequent DC generation is poorly documented. The method used to isolate monocytes may have an impact on the subsequent function of DC by affecting their ability to express costimulatory molecules (CD80/86), maturation marker (CD83) and/or to produce important immunomodulatory cytokines. In this study, we show that the positive selection of monocytes by anti-CD14-coated microbeads inhibits the lipopolysaccharide (LPS)-induced production of interleukin (IL)-12, IL-10 and tumour necrosis factor-alpha (TNF-alpha) from human DC. However, when DC were grown from monocytes isolated by plastic adherence, LPS induced the production of much higher levels of these cytokines. DC derived from adherence-isolated monocytes induced the development of potent cytotoxic T lymphocytes of the Tc1 subset specific for influenza matrix protein, as confirmed by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT), cytotoxicity assay, major histocompatibility complex (MHC)-peptide tetrameric complexes and T helper 1/T helper 2 (Th1/Th2) cytokine production assays.