Tropomyosin has been isolated from microvilli preparations from 13762 rat mammary adenocarcinoma ascites tumor cells by Triton extraction and pelleting of the microvillar microfilament core, extraction of the microfilament core with 1 M KCl, heat treatment, and hydroxyapatite chromatography. Three major isoforms, designated 31K-a (acidic), 31K-b (basic), and 29K, were identified as tropomyosins by two-dimensional isoelectric focusing-dodecyl sulfate electrophoresis, a urea shift on dodecyl sulfate electrophoresis, chemical cross-linking, amino acid analysis, and molecular weight determinations. The native (60,000) and subunit (31,000 and 29,000) molecular weights, the amino acid composition, and the stoichiometry for binding to F-actin (actin/tropomyosin, 6:1) were typical of nonmuscle tropomyosins. The amount of tropomyosin present in the microvilli preparations is sufficient to saturate about half of the microvillar F-actin. By two-dimensional isoelectric focusing-dodecyl sulfate electrophoresis, the 31K isoforms appeared similar to isoforms of normal rat kidney cells but the 29K isoform was apparently smaller than any normal rat kidney isoforms. All three isoforms bound to F-actin, but the 29K form bound most strongly. Its behavior was similar to that of muscle tropomyosin, exhibiting saturable binding as a function of both ionic strength and Mg2+ concentration. In contrast, the 31K isoforms bound more weakly and required higher concentrations of Mg2+ for binding than that required for saturation with 29K (4 mM). These results clearly indicate that nonmuscle tropomyosin isoforms from a single source and location (subplasmalemmal) in the cell can exhibit different properties.