We grew loblolly and ponderosa pine seedlings in a factorial experiment with two CO2 partial pressures (35 and 70 Pa), and two nitrogen treatments (1.0 and 3.5 mol m−3 NH4+), for one growing season to examine the effects of carbon and nitrogen availability on leaf construction cost. Growth in elevated CO2 reduced leaf nitrogen concentrations by 17 to 40%, and increased C:N by 22 to 68%. Elevated N availability increased leaf N concentrations and decreased C:N. Non-structural carbohydrates increased in high-CO2-grown loblolly seedlings, except in fascicles from low N, and in ponderosa primary and fascicle leaves grown in high N. In loblolly, increases in starch were nearly 2-fold greater than the increases in soluble sugars. In ponderosa, only the soluble sugars were affected by CO2. Leaf construction cost (g glucose g−1 dm) varied by 9.3% across all treatments. All of the variation in loblolly leaf construction cost could be explained by changes in non-structural carbohydrates. A model of the response of construction cost to changes in the mass of different biochemical fractions suggests that the remainder of the variation in ponderosa, not explained by non-structural carbohydrates, is probably attributable to changes in lignin, phenolic or protein concentrations.