Direct intratumoral introduction of therapeutic or regulatory genes is a developing technology with potential application for cancer gene therapy. Macrophage inflammatory protein-1 beta (MIP-1beta) is a chemokine which can chemoattract immune cells such as T cells. In the present study, murine colorectal adenocarcinoma CT26 cells were transfected with a recombinant adenovirus (AdhMIP-1beta) carrying the human MIP-1beta gene. 24 h post-transfection, hMIP-1beta levels reached approximately 980 pg/ml in supernatants of 10(6) hMIP-1beta-transfected CT26 cells. Moreover, the supernatants exhibited chemotactic activity for CD8(+) T cells, CD4(+) T cells, NK cells and immature DCs. Intratumoral injection of AdhMIP-1beta significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice. Intratumoral hMIP-1beta gene transfer also induced powerful tumor-specific CTL responses in vivo. The therapeutic effects of hMIP-1beta gene therapy were greatly reduced following in vivo depletion of both CD4(+) and CD8(+) T cells, but were unaffected by depletion of single T cell subsets. Immune cell depletion experiments also revealed that NK cells played an important role in hMIP-1beta-induced antitumor responses. These results suggest that intratumoral expression of hMIP-1beta has the potential effect to induce host antitumor immunity and may prove to be a useful form of cancer gene therapy.