S100A1 is a new regulatory protein of myocardial contractility that is differentially expressed in early and late stages of myocardial hypertrophy. In order to further investigate the multiple functions of S100A1 in various assay systems we developed a new strategy for isolating biologically active S100A1 protein. After EDTA extraction of myocardium or recombinant bacteria, S100A1 was purified by Octyl-Sepharose hydrophobic interaction chromatography and HiTrapQ anion-exchange chromatography yielding 1.4-2.0 mg/100 g wet tissue and 0.7-1.0 mg/100 ml bacterial culture. Native porcine as well as human recombinant S100A1 revealed biological activity in physiological and biochemical assays.