OBJECTIVE: L-Glutamine, L-arginine, RNA, and omega-3 polyunsaturated fatty acids (PUFAs) have been incorporated into nutritional formulas to improve immunity of patients with gastrointestinal cancer. We therefore examined the individual and net effects of these immunonutrients on four different human colorectal adenocarcinoma cell lines. METHODS: LS174T, HT-29, CO112, and Caco-2 cells were exposed to dilutions of 1:50, 1:100, and 1:1000 of a mix or individual components of a mix of 15 g/L of L-glutamine, 16.3 g/L of L-arginine, 1.6 g/L of RNA, and 2.7 g/L of omega-3 PUFAs. Cell growth kinetic was assessed using cell count with a flow cytometer. Cell cycle and apoptosis were evaluated with double fluorescence-activated cell sorter analyses using bromodeoxyuridine labeling index and annexin V staining, respectively. One-way analysis of variance and Student's t tests were used for comparison. RESULTS: Evaluation of the cell growth kinetic over an 18-d period showed that the immunonutrient mix stimulated cancer cell growth only when diluted > or =100 times. Individual component evaluation indicated that the cell growth stimulation was mainly due to the presence of L-glutamine and to a lesser extent RNA in the mix. L-Arginine had no effect. At a lower dilution of 1:50, omega-3 PUFA concentrations were sufficient to induce cell cycle arrest and massive cell death in part through apoptosis. CONCLUSION: These results suggest that cancer cell growth stimulation by current immunonutrient formulas is unlikely due to predominant cytotoxic effect of omega-3 PUFAs.