G207 is a multi‐mutated, replication‐competent type‐1 herpes simplex virus designed to target, infect, and lyse neurological tumors. This study examines the feasibility of using G207 in the treatment of human colorectal cancer and defines the biological determinants of its antitumor efficacy. This virus was tested on five human colorectal cancer cell lines in vitro to determine efficacy of infection and tumor cell kill. These results were correlated to measures of tumor cell proliferation. In vivo testing was performed through direct injections of G207 into xenografts of human colorectal cancer tumors grown in flanks of athymic rats. To evaluate an alternate method of administration, hepatic portal vein infusion of G207 was performed in a syngeneic model of liver metastases in Buffalo rats. Among the five cell lines tested, infection rates ranged between 10% and 90%, which correlated directly with S‐phase fraction (8.6%–36.6%) and was proportional to response to G207 therapy in vitro (1%–93%). Direct injection of G207 into nude rat flank tumors suppressed tumor growth significantly vs. control (0.58 ± 0.60 cm³ vs. 9.16 ± 3.70 cm³, P<0. 0001). In vivo tumor suppression correlated with in vitro effect. In the syngeneic liver tumor model, portal infusion resulted in significant reduction in number of liver nodules (13 ± 10 nodules in G207‐treated livers vs. 80 ± 30 nodules in control livers, P<0.05). G207 infects and kills human colorectal cancer cells efficiently. In vitro cytotoxicity assay and tumor S‐phase fraction can be used to predict response to treatment in vivo. This antineoplastic agent can be delivered effectively by both direct tumor injection and regional vascular infusion. G207 should be investigated further as therapy for colorectal cancer and liver metastases.—Kooby, D. A., Carew, J. F., Halterman, M. W., Mack, J. E., Bertino, J. R., Blumgart, L. H., Federoff, H. J., Fong, Y. Oncolytic viral therapy for human colorectal cancer and liver metastases using a multi‐mutated herpes simplex virus type‐1 (G207). FASEB J. 13, 1325–1334 (1999)