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-1β) 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-1β) carrying the human MIP-1β gene. 24 h post-transfection, hMIP-1β levels reached approximately 980 pg/ml in supernatants of 106 hMIP-1β-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-1β significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice. Intratumoral hMIP-1β gene transfer also induced powerful tumor- specific CTL responses in vivo. The therapeutic effects of hMIP-1β 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-1β-induced antitumor responses. These results suggest that intratumoral expression of hMIP-1β has the potential effect to induce host antitumor immunity and may prove to be a useful form of cancer gene therapy.