Quantitative amino acid analysis by gas chromatography was applied to the determination of 4-hydroxy-L-proline in biological material. After addition of a known amount of allo-4-hydroxy-D-proline, quantitative analysis of 4-hydroxy-L-proline can be performed by gas chromatographic separation of the amino acid enantiomers; since the standard and sample have identical chemical behaviour, they respond identically during derivatization. The accuracy, reproducibility, and sample requirement of this procedure are more advantageous than those of hitherto existing methods. This study is also an example of the application of chiral stationary phases, which permit the simultaneous separation of amino acids and amino acid enantiomers.