Current biofuel feedstock crops such as corn lead to large environmental losses of N through nitrate leaching and N 2O emissions; second-generation cellulosic crops have the potential to reduce these N losses. We measured N losses and cycling in establishing miscanthus ( Miscanthus * giganteus), switchgrass ( Panicum virgatum L. fertilized with 56 kg N ha -1 yr -1), and mixed prairie, along with a corn ( Zea mays L.)-corn-soybean [ Glycine max (L.) Merr.] rotation (corn fertilized at 168-202 kg N ha -1). Nitrous oxide emissions, soil N mineralization, mid-profile nitrate leaching, and tile flow and nitrate concentrations were measured. Perennial crops quickly reduced nitrate leaching at a 50-cm soil depth as well as concentrations and loads from the tile systems (year 1 tile nitrate concentrations of 10-15 mg N L -1 declined significantly by year 4 in all perennial crops to <0.6 mg N L -1, with losses of <0.8 kg N ha -1 yr -1). Nitrous oxide emissions were 2.2 to 7.7 kg N ha -1 yr -1 in the corn-corn-soybean rotation but were <1.0 kg N ha -1 yr -1 by year 4 in the perennial crops. Overall N balances (atmospheric deposition+fertilization+soybean N 2 fixation-harvest, leaching losses, and N 2O emissions) were positive for corn and soybean (22 kg N ha -1 yr -1) as well as switchgrass (9.7 kg N ha -1 yr -1) but were -18 and -29 kg N ha -1 yr -1 for prairie and miscanthus, respectively. Our results demonstrate rapid tightening of the N cycle as perennial biofuel crops established on a rich Mollisol soil.