Docosahexaenoic acid (DHA), upon incorporation into tumor tissue, has the potential to sensitize tumors to the effects of chemotherapy or radiation therapy. Although DHA has usually been supplied to tumor tissue in the diet, appropriate dietary conditions required to obtain optimal tumor levels have not been established. Hence, we studied mammary tumor tissue responses in rats fed various durations and doses of DHA. Rats fed a palm oil enriched diet (diet 0) were switched to diets providing either 0.8 g DHA/day (diet 1) or 1.5 g DHA/day (diet 2). Tumor tissue fatty acid composition was analysed at baseline (diet 0), at weeks 1, 4 and 9 during diet 1 and at week 4 during diet 2. Dietary DHA supplementation differentially increased DHA within phospholipids (PL) and triacylglycerol (TAG) fractions in tumors. DHA level equilibrated between 2 and 4 weeks in PL while DHA increase was more progressive in TAG and did not reach a steady state. A higher dose of DHA further increased DHA content in tumor PL and TAG (P = 0.018 and P < 0.001, respectively). DHA concentration in plasma PL was positively correlated with DHA in tumor PL (r = 0.72; P = 0.0003) and TAG (r = 0.64; P = 0.003). We conclude that dietary DHA supplementation enhances tumor content of DHA in a time- and dose-dependent manner, and that the DHA level in plasma PL could be used as a proxy for tumor DHA. These findings have implications for dietary DHA supplementations in cancer patients.