BACKGROUND: Recent reports have described a new strategy for differentiation and maturation of monocyte (Mo)-derived DCs within only 48 h of in vitro culture (fast-DC). Here we assess the efficacy of the fast-DC to process and present different Aspergillus fumigatus and CMV Ag preparations to autologous T cells, compared with DCs generated in standard 7-day cultures (standard-DC). METHODS: Adherent blood Mo were treated with GM-CSF and IL-4 (1 day for fast-DC, 5 days in the standard-DC) to generate immature DCs, and then were matured for either 1-2 days (fast-DC) or 2 days (standard-DC) with inflammatory cytokines. DCs were pulsed with A. fumigatus or CMV Ag preparation immediately prior to maturation, or infected after maturation with adeno-pp65. Mature DCs were then used to prime Ag-specific proliferative and cytotoxic T lymphocytes (CTL) responses. RESULTS: Fast-DC were CD14- and expressed mature DC surface markers to the same degree as standard-DC, and maintained this phenotype after withdrawing cytokine from the cultures. Fast-DC and standard-DC were equally capable of inducing A. fumigatus and CMV-specific T-cell proliferation, as well as priming Ag-specific CTL activity. The Aspergillus- and CMV-specific CTL were of mixed CD3+/CD4+ and CD3+/CD8+ phenotype, and specifically killed autologous DC pulsed with A. fumigatus Ag and autologous CMV infected fibroblasts, respectively. DISCUSSION: Fast-DC are as effective as standard-DC in the generation of Ag-specific T-cell responses. Moreover, use of fast-DC not only reduces labor and supply cost, as well as workload and time, but also increases the number of DCs derived from adherent Mo, which may facilitate the use of DCs in clinical trials of cellular immunotherapy.