We report a method of isolation of aldosterone and tetrahydroaldosterone from urine and undiluted plasma which is based on extraction with Amberlite XAD-2 resin and purification by low pressure liquid chromatography using microbore LH-20 columns. Thin-layer chromatography on polyamide plates is introduced as the final step of a procedure designed to reduce destruction of steroids to a minimum, and to provide, in addition, a facility for multisample processing. Using a synthetic substrate, it has been shown that enzyme hydrolysis of steroid conjugates is possible whilst still adsorbed to the resin after extraction although the yield is lower than when hydrolysis is performed in solution.