We developed a single-stage technique involving a proteolytic enzyme (pepsin) for the solubilization of cardiac muscle to measure magnesium in tissue. This new method has been developed specifically for use with very small cardiac biopsy samples (less than 1 mg) obtained with modern myocardial biopsy forceps. Pepsin digestion of the tissue releases magnesium ions into solution and, after centrifugation and dilution with lanthanum chloride, the resulting supernate is suitable for analysis by atomic absorption spectrometry. Recovery of magnesium after a single digestion approaches 99%. In direct comparison studies of pepsin digestion and a traditional method involving nitric acid extraction, the pepsin digestion consistently yielded more magnesium. In contrast to traditional methods of tissue solubilization, pepsin digestion is well suited to extraction of magnesium from the small biopsy samples commonly presented for analysis in clinical practice.