Except for albumin, a 41,000-dalton protein (41K) in the cytosol of the Dunning R-3327 rat prostatic adenocarcinoma was found to be the most abundant soluble protein. This protein was purified in nearly homogeneous state by conventional chromatographies. After the first chromatography, because of the adhesive nature of the protein, 0.5% SDS and 2 M urea were necessary for subsequent steps of purification. The amino acid composition of the purified 41K was similar to that of actin isolated from rabbit skeletal muscle. Alternatively, 41K could be extracted from the Dunning tumor in the presence of ATP and dithiothreitol, under conditions in which actin molecules are depolymerized, and could be purified by the same method as cytoskeletal actin. The purified protein showed properties similar to rat skeletal muscle actin in amino acid composition and antigenicity. Both 41K proteins were found to be composed of four components having different isoelectric points. These results indicate that most actin exists in a depolymerized form as a cytosol protein of 41,000 daltons in the Dunning tumor and is composed of at least four isomers.