"Naphthenic acids" comprise a group made up of hundreds of chemically and structurally related alkanoic compounds, which are natural components of bitumen. They are believed to be the main cause of acute toxicity of bitumen processing wastewaters, but also have commercial applications. The naphthenic acids distributions of extracts from several Athabasca oil sands ores, tailings ponds, as well as commercial mixtures were analyzed by gas chromatography-mass spectrometry of their t -butyldimethylsilyl derivatives. Statistical methods for comparing the resulting three-dimensional fingerprints were developed. An HPLC method was adapted for analysis of naphthenic acids concentrations in laboratory cultures. The biodegradation of Kodak salts and Merichem acids was characterized by measuring the rate of mineralization, naphthenic acids loss, toxicity decrease, and changes in component distribution.