This assay was carried out in order to evaluate the effect of the culture medium N content on gas production and on apparent organic matter digestibility prediction of grass silage. To carry out this experiment, 24 grass silage samples of known apparent organic matter digestibility were selected from the collection belonging to the Centro de Investigacións Agrarias de Mabegondo. Samples were incubated in buffered rumen fluid supplemented and non supplemented with nitrogen and the obtained cumulative gas production was adjusted to a generalized Michaelis-Menten model. Culture medium and sample nitrogen content were not high enough to allow an optimal microbial growth and activity (16.7 vs. 23.1 h to obtain the half of potential gas production with and without nitrogen suplementation; P < 0.001). As a consequence, the correlations between the gas production parameters obtained with a non supplemented medium and the chemical constituents or the apparent organic matter digestibility were worse than those obtained with a supplemented culture medium. All these results led to a worse apparent organic matter digestibility predictive equation when a non supplemented nitrogen culture medium was used (r2 = 0.519) compared with those obtained with supplemented medium (r2 = 0.758).