Natural gas is generated throughout the burial history of sedimentary rocks. Three principal stages of gas generation are recognized. In the first stage, anaerobic bacteria reduce carbon dioxide to form methane during the earliest, low-temperature stage. Later, methane is generated along with other gaseous and liquid hydrocarbons during the thermochemical transformation of organic matter to petroleum. Finally, methane is formed as a stable residual end-product when increased burial temperatures result in the thermal destruction of organic matter. The gas formed during each of these stages has a characteristic chemical composition and stable carbon isotope ratio of methane. Bacterial gas is predominantly methane that is isotopically light (δ¹³C = −90 to −50 per mil). Methane originating during the thermal generation of petroleum is always accompanied by ethane and heavier hydrocarbons, and is isotopically heavier (δ¹³C = −55 to −35 per mil). Natural gas formed as a result of prolonged high temperatures becomes increasingly devoid of heavier hydrocarbons, but has higher contents of inorganic gases (CO₂, H₂S, N₂). The isotopic composition of methane produced in this final stage approaches that of the parent organic matter (δ¹³C = −30 to −25 per mil). Natural gas accumulations show a continuous gradation of chemical and isotopic compositions between these characteristic end-member types. Three major factors produce these gradations:

1. mixing of gases of different origins;

2. temperature-dependent isotopic fractionation; and

3. source-dependent chemical fractionation.

For any given gas accumulation it may be difficult to determine the controlling factors on the basis of gas analysis alone. Consequently, a knowledge of the chemical nature of the organic matter and of the geologic history of a source bed is needed. Examples of gas accumulations and the relative importance of mixing vs. fractionation, as they relate to isotopic and chemical composition, are illustrated with examples from the Cook Inlet, Alaska; Texas-Louisiana offshore, Gulf of Mexico; Cretaceous of the northern Great Plains; and Devonian black shales of the Appalachian basin.