We describe the isolation, using preparative capillary gas chromatography, of hydrocarbon fractions from an unresolved complex mixture (UCM) of hydrocarbons isolated from a biodegraded crude oil. Some of the individual hydrocarbons in these fractions were then resolved by gas chromatography (GC) and identified using GC–mass spectrometry (GC–MS). The mass spectra contained distinct molecular ions. As a preliminary example, after preparative open column chromatography, preparative HPLC (three columns in series) and preparative GC on two stationary phases, one of the compounds from the UCM was tentatively identified using electron impact ionisation GC–MS as a novel C₂₆ 17-desmethyl triaromatic steroid. If the concentration of this compound (ca. 4 μg g⁻¹ whole oil) is typical of other individual compounds in the UCM, the proportion of UCM represented by each compound is obviously very small (<0.0004%) and suggests that the whole oil UCM contains in the order of 250,000 mostly unidentified compounds. This suggests that oil UCMs are amongst the most complex mixtures of organic compounds on Earth and explains the difficulty of UCM component identification. Nevertheless, it also indicates that multi-step chromatography can lead to the identification of individual compounds, some of which have geochemical, petrochemical and environmental significance. Acquisition of such knowledge is fundamental if we are to improve our understanding of petroleum.