A three-dimensional simulation of gravitational separation, defined as the process of atmospheric molecule separation under gravity according to their molar masses, is performed for the first time in the upper troposphere and lower stratosphere. We analyze distributions of two isotopes with a small difference in molecular mass ( 13 C 16 O 2 (M i = 45) and 12 C 16 O 2 (M i = 44)) simulated by the National Institute for Environmental Studies (NIES) chemical transport model with a parameterization of molecular diffusion. The NIES model employs global reanalysis and an isentropic vertical coordinate and uses optimized CO 2 fluxes. This study includes a comparison with measurements recorded by cryogenic balloon-borne samplers in the lower stratosphere and two-dimensional model simulations. The benefits of the NIES TM simulations are discussed. We investigate the processes affecting gravitational separation by a detailed estimation of terms in the molecular diffusion equation. At the same time, we study the age of air derived from the tracer distributions. We find a strong relationship between age of air and gravitational separation for the main climatic zones. The advantages and limitations of using age of air and gravitational separation as indicators of the variability in the stratosphere circulation are discussed.