Biochar amendments can modify fertilizer nitrogen (N) availability in soil and crop N uptake. However, how biochar addition affects crop N uptake and fertilizer N recovery under various N levels is not yet well understood. To address this question, we conducted a two-season [cotton (Gossypium hirsutum L.)-barley (Hordewn vulgare L.) rotation] pot experiment that included four N fertilizer rates (0, 75, 150, and 300 kg N ha(-1), supplied as urea-N-15) combined with two straw-biochar rates (0 and 15 t ha(-1)). Soil properties, plant root morphology, N uptake, and biomass yield were studied. Biochar addition decreased soil inorganic N content but increased ureaN retention at cotton harvest, leading to 32% of the applied urea-N accumulating in soil compared with 27% without biochar, averaged across fertilizer N rates. Use of N-15 fertilizer showed that biochar increased plant uptake of indigenous soil N, not fertilizer N. An obvious decrease in urea-N-15 recovery induced by biochar was observed at 75 kg N ha(-1), but not at 150 or 300 kg N ha(-1). The efficiency of urea-N-15 recovery by plants ((NRE)-N-15, 34-45%), measured using the tracer method, was much lower than that measured using the traditional nonisotope method (NRE, 67-96%). At barley harvest, 2-5% of the urea-N, applied in the first season, was taken up by plants, and 12-19% remained in soils receiving biochar. We concluded that straw-biochar addition increased soil N-15 fertilizer retention and plant N uptake of indigenous soil N, not fertilizer N, and the increased indigenous soil N uptake persisted into the second cropping season.