Natural gas is considered as the most environmentally acceptable fossil fuel, thus it is an alternative to petroleum derivate, dominantly used in internal combustion engines. Physical properties of a fuel as well as behavior during combustion process are affected by its composition. This article presents analysis of effect of natural gas composition on methane number (MN), conducted for natural gas deposits in Serbia. For empirically determination of MN: California air resources board (CARB) method and Linear and Anstalt fur Verbrennungskraftmaschinen list (AVL) method have been applied. Effect of reactive hydrogen and carbon ratio, Wobbe index and content of inert matter on MN is discussed. For compositions characterized with total share of inert compounds higher than 5%, impact of inert components becomes significant, without observed dependency between reactive hydrogen and carbon and MN. An analysis has shown that carbon dioxide and nitrogen have differently affect MN. Carbon dioxide affects the increase of MN due to higher molar specific heat, appearance of dissociation, and higher activation energy.