The need for biochemical quantities of nonmuscle actin has been increased by observations that actin isoform composition of a cell influences the cell's motile and structural properties. In addition, the number of actin binding proteins that exhibit different binding interactions with beta- and gamma-actin compared to alpha-actin from skeletal muscle is growing. We report a procedure designed to purify actin from nonmuscle tissues employing extraction of monomeric actin from tissues with high concentrations of Tris, chromatography on DE-53 cellulose, and affinity chromatography of DNase I-agarose. The preparation is easy to perform and yields quantities of nonmuscle actin sufficient for biochemical and cell biological assays. Actin from bovine erythrocytes and from brains of adult and embryonic chickens was obtained using this method, which can be readily used with other sources of tissue. Coomassie-Blue-stained SDS gels of the purified actin show no contaminants; capping protein, a common contaminant of actin preparations, is absent by immunoblotting. This method for purifying nonmuscle actin will be useful to investigate functional differences in the biology of actin isoforms or their regulating proteins.