Actin and four major associated proteins have been identified in the microvillus cytoskeleton from chicken intestinal brush borders isolated in the presence of Ca2+-chelating agents. We have isolated microvilli from rabbit small intestine in the presence of Ca2+ and found by sodium dodecyl sulfate-gel electrophoresis that a protein of 33,000 daltons exists in a molar ratio to actin of 1:3 within the isolated microvillus. The protein could be extracted from acetone powder of microvilli in the absence, but not in the presence, of Ca2+. Thus, for its purification the acetone powder was extracted twice with buffer containing Ca2+ and then with buffer containing a Ca2+-chelator. The 33,000-dalton protein was purified from the last extract by Sephadex G-100 gel filtration and DEAE-cellulose column chromatography. The purified protein was a water-soluble monomeric protein with a molecular weight of 33,000. Antisera against the purified protein raised in guinea pigs reacted with the antigen but not with actin or tropomyosin purified from rabbit skeletal muscle. Freezing-thawing resulted in aggregation of the purified protein, and the aggregates showed filamentous and sheet-like structures. These results suggests that the 33,000-dalton protein is a major component of the microvillus cytoskeleton.