The applicability of high-performance hydrophobic interaction chromatography using newly developed silica-based ether-bonded phases is demonstrated in the purification of the rat liver enzymes carbamoylphosphate synthetase I and ornithine transcarbamoylase from crude mitochondrial extracts. As a result of the mild adsorption/elution conditions in this high-performance chromatographic mode, the enzymes are recovered in 20 min with 3- to 15-fold increases in specific activity. Since the enzymes are labile and may aggregate in solution, in one case up to Mr 330,000, this rapid purification demonstrates the potential of hydrophobic interaction chromatography in complex biological systems.