A new and versatile method for the identification and quantification of short-chain fatty acids, such as formic, acetic, propionic, butyric, isobutyric, valeric, isovaleric and mercaptoacetic acids, in biological specimens by high-performance liquid chromatography is described. After sample purification by vacuum transfer and concentration by alkaline freeze-drying, the acids were measured without any further preparative step, using a sulphonated polystyrene-divinylbenzene column as stationary phase. Ultraviolet detection of the native acids was done at 214 nm. Peak resolution and reproducibility were as good as with gas chromatography. Many examples of the application of this method to a variety of biological specimens and fluids both from the rats and humans are described.