Given the great potential value of stable isotopes in a variety of scientific investigations, surprisingly little attention has been paid to the underlying physiological and biochemical mechanisms that account for trophic increases in delta(15)N values. This has lead to a general call for controlled studies investigating the relationship between organismal diet and corresponding isotopic composition. We conducted a series of laboratory studies varying dietary nitrogen content and measuring corresponding variations in organismal delta(15)N values. Specifically, we investigated the relationship between the delta(15)N values of the anomopod crustacean, Daphnia magna, and the C:N ratio of its food, the green algae, Scenedesmus acutus. Daphnids were raised to a standard life stage on three types of S. acutus as food, which ranged in C:N (atomic) from 7.3 to 24.8. The average C:N of the daphnids was 6.0. N-15 enrichment was found to be strongly linearly related to the C:N of the algae, ranging from nearly zero to approximately 6 parts per thousand, which would normally be considered a span of almost two trophic levels. The delta(15)N values of the daphnids and the diet-tissue isotope fractionation factor (Delta(dt) = D. magna delta(15)N - S. acutus delta(15)N), were inversely related to the nitrogen content of the algae (R-2 = 0.82 and 0.99 in two separate runs). To our knowledge, this is the first controlled study of nitrogen balance and delta(15)N values in animals.