The aim of this study was to quantify the spatial distribution of the net photosynthesis of a multi-layer crop. A multi-layer model (FORUG) was used to simulate the crop photosynthesis of tomato ( Lycopersicon esculentum Mill.) in the three-dimensional space inside a plant growth chamber. The model inputs were, on the one hand photosynthetic characteristics (maximal photosynthetic rate, quantum efficiency and respiration rate) determined at leaf level, and the microclimatic data on the other hand. Scaling-up to the level of the crop was done, taking into account the spatial distribution of leaf area index (LAI), leaf angle (or extinction coefficient), air temperature and photosynthetically active radiation (PAR). The simulated output was the net carbon exchange at a specific time step or the cumulative net carbon exchange over a specific period of time. Inside the test chamber spatial temperature differences ranged from 22.4 up to 32.9 °C at an inlet temperature of 22 °C and the lights on. Light intensity decreased gradually within the crop, resulting in very low light intensities at the bottom layers of the climate chamber. This resulted in the observation that the respiration rate was not compensated at low light intensities. The simulated photosynthesis of the 32 ‘cells’ of the test chamber ranged from −1.12 up to 5.94 μmol CO 2 m −2 s −1. From a sensitivity analysis of the model at low (20% of maximum) and high (80% of maximum) light intensities, it was concluded that the spatial distribution of light, air temperature and LAI should be well known, just as the parameters describing the light response of the photosynthesis process. Moreover, this spatial distribution should be accurately taken into account.