BACKGROUND We hypothesized that interferon alpha (IFN-alpha) and unmethylated cytosine-phosphothioate-guanine (CpG)-rich oligodexoynucleotides (CpG ODNs) would elicit potent antitumor activity due to the ability of this treatment combination to activate complimentary signal transduction intermediates. METHODS Peripheral blood mononuclear cells treated with CpG ODNs, IFN-alpha, or both agents combined were evaluated for cytotoxicity against human melanoma cells, Jak-STAT signal transduction by flow cytometry, and ISG-15 gene expression by real-time polymerase chain reaction. The effects of CpG ODNs and IFN-alpha were evaluated in murine models of melanoma in wild-type, IFN-gamma-deficient, and STAT1-deficient mice. Negative controls in all experiments included treatment with control ODN or phosphate-buffered saline. RESULTS Treatment of peripheral blood mononuclear cells with a combination of CpG ODNs and IFN-alpha resulted in enhanced cytotoxicity, activation of natural killer cells, IFN-alpha-induced STAT1 phosphorylation, and transcription levels of ISG-15. These immunostimulatory effects of CpG ODNs were associated with increased expression of STAT1 and STAT2 proteins. Administration of CpG ODNs plus IFN-alpha elicited superior antitumor activity in a murine model of B16 melanoma compared with either agent alone. The antitumor properties of CpG ODNs were dependent on STAT1-mediated signal transduction within the host but independent of endogenously produced IFN-gamma. CONCLUSIONS CpG ODNs represent potent immune stimulants that elicit antitumor effects through STAT1-mediated signal transduction.