The objective of this study was to determine whether mesquite (Prosopis velutina) seedlings have a preference for the ammonia or nitrate form of nitrogen (N), and to determine the optimum rate of N to maximize growth and minimize N leaching when seedlings are grown in different substrates. Mesquite seedlings were fertigated with different ratios of NH4+: NO3- to determine effects on shoot and root growth and N-uptake efficiency. Nutrient solution containing 67% NH4+ : 33% NO3- resulted in greatest biomass after 120 days of fertigation. N leachate remained stable until 12 weeks after the onset of treatment, but increased significantly by week 16. Subsequently, mesquite seedlings were grown in sand or soilless media and were fertigated with a solution of 67 % NH4+: 33% NO3- at a rate of 25, 50, 100, or 200 mg·L-1 of N. After 60 days, plants in media produced 41% more leaves and total biomass compared to those in sand. Leaf number was greatest for plants grown at 200 mg·L-1 of N in both substrates. Root biomass of plants in media showed no response to increasing N concentrations while root biomass of seedlings in sand were similar for the three lower N concentrations and nearly doubled for the highest one. Shoot biomass of seedlings receiving 25, 50, or 100 mg·L-1 of N was similar, but more than doubled for plants fertigated with 200 mg·L-1 of N. N leachate losses were highest from seedlings growing in sand and receiving the two higher N fertigations, those in media had greatest N leachate loss when fertigated at 200 mg·L-1 of N. For balanced mesquite seedling growth and minimum N leaching losses, concentrations between 50 to 100 mg·L-1 of N are recommended. Implications of using a sand culture system vs. soilless growing substrate for nutrition studies will be discussed.