The organization of microvillus actin and its associated proteins have been investigated in sublines of mammary ascites tumors (MAT) with mobile (MAT-B1) and immobile (MAT-C1) cell surface receptors. Microvilli isolated from these sublines differ in morphology (branched for MAT-C1 versus unbranched for MAT-B1) and the presence of a 58,000-dalton polypeptide (58K). 58K is found associated with MAT-C1 microvilli, microvillar cytoskeletons obtained by nonionic detergent extractions, and microvillar membranes prepared under conditions which depolymerize actin microfilaments. By extraction with actin-stabilizing buffers (isotonic Triton-Mg-ATP) microvillar actin can be fractionated into four forms. About 40% of the actin is sedimented at low speed (7,500g, 15 min). The pellets contain microfilaments; actin and alpha-actinin are the predominant proteins. High-speed pellets from these low-speed supernates contain about 10% of the actin as a transmembrane complex with a cell surface glycoprotein (cytoskeleton-associated glycoprotein, [CAG] 75-80,000 daltons) in MAT-B1 cells or with CAG and 58K in MAT-C1 cells. Transmembrane complexes can be purified from MAT-B1 and MAT-C1 microvillar membranes in Triton-containing buffer by gel filtration or sucrose density gradient centrifugation. The presence of only CAG and actin in the MAT-B1 transmembrane complex strongly suggests the direct interaction of actin and a cell surface component. The high-speed supernates contain soluble actin. By gel filtration or rate-zonal sucrose density gradient centrifugation about 30% of the microvillar actin is found as small oligomers and about 10% as G-actin in this extraction buffer. We suggest that the actin-containing transmembrane complexes may serve as membrane-association sites for oligomeric actin segments and microfilaments and as initiation sites for actin polymerization.