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