Oxidation of free fatty acids (FFA) by Ehrlich ascites tumor in mice was studied in vivo by the direct measurement of 3H2O formed from [9,10-(3)]palmitate. The FFA tracer complexed to serum albumin was injected i.p. into unanesthetized mice, and blood plasma 3H2O was measured at different time points for 30 min. The contribution of 3H2O by desaturation of labeled palmitate to monounsaturated fatty acids in the tumor was estimated by the use of [1-14C]palmitate and was shown to be negligible during the course of our experiments. In order to estimate the rates of tumor FFA oxidation, the kinetics of the tumor-host water distribution system was studied by injecting tracer 3H2O i.p. and following the disappearance of 3H2O in the blood plasma at different time points for 30 min. The results of these experiments were used to compute the tumor FFA oxidation rate by multicompartmental analyses and SAAM. Despite the nearly anaerobic state of the ascites tumor fluid in vivo, cancer cells suspended in this fluid oxidized FFA at least as fast as they do in vitro under aerobic conditions. Moreover, according to our current estimate, the need of the tumor for FFA as a metabolic fuel appears to be much greater than its net lipid needs for growth.