This study was designed to investigate possible additive or synergistic action among sphingomyelin (SPH), cis-9,trans-11-conjugated linoleic acid (CLA), and butyrate (BTY) against colon cancer and modulation of immune functions in vivo in male Sprague-Dawley rats. Each of the 5 groups of rats was fed either 35 mg SPH, 100 mg CLA, or 100 mg BTY/kg body weight, a combination of the 3 compounds at the same doses, or none of the compounds, for 7 wk. Rats were injected with azoxymethane, a colon carcinogen, to induce the formation of aberrant crypt foci, preneoplastic lesions of colon cancer. Parameters measured included number and multiplicity (number of crypts per focus) of aberrant crypts, immune functions such as innate immunity (natural killer cell cytotoxicity), humoral immunity (development of antibodies), and cell-mediated immunity (delayed-type hypersensitivity). Results show that the groups treated with SPH, CLA, and BTY individually had significantly higher natural killer cell (NK) cytotoxicity than the group treated with all compounds. The CLA group also had significantly higher NK activity than the control group. This study shows that the three compounds may not act additively or synergistically either to inhibit the development of aberrant crypts or to enhance immune functions. In fact, exposure to the combined compounds may be antagonistic to enhancement of NK function by the individual chemicals.