Knowledge of the fractionation of nitrogen isotopes by phytoplankton is a key requirement for the calibration of the new delta(15)N paleotracer. An essential part of information required in this calibration concerns the magnitude of isotopic fractionation during the incorporation of N substrates by phytoplankton. To this end, the delta(15)N of batch cultures of Thalassiosira pseudonana grown on nitrate, nitrite, ammonium, and urea was determined. This paper reports the first delta(15)N study of phytoplankton growth on urea (e.g. organic N substrate). The delta(15)N of the particulate nitrogen (PN) collected during the logarithmic growth phase, thus for N-sufficient cells, was lower than the delta(15)N of the source due to kinetic isotope fractionation. With increasing drawdown of the N substrate, the delta(15)N of the accumulating PN increased in accordance with the Rayleigh distillation model. Enrichment factors (epsilon) derived from a least-squares analysis of the accumulated delta(15)N(PN) data were 5.2 +/- 0.2 parts per thousand, 0.9 +/- 0.6 parts per thousand, 20 +/- 1 parts per thousand, and 0.8 +/- 0.6 parts per thousand for NO(3)(-), NO(2)(-), NH(4)(+), and urea incorporation, respectively. Overall, epsilon values for nitrate incorporation were consistent with field estimates of similar to 6 parts per thousand, and could be used to estimate past relative nitrate utilization as estimated from the delta(15)N of bulk sedimentary organic matter.