Tropomyosin is a key component of the contractile systems found in muscle and nonmuscle cells of higher eukaryotes. Based on properties common to all tropomyosins, we have purified a protein from Saccharomyces cerevisiae that resembles tropomyosins from higher cells. The yeast protein remains soluble after heat treatment at 90 degrees C, has an apparent polypeptide molecular weight of 33,000, an isoelectric point of 4.5, a Stokes radius of 3.5 nm, and a sedimentation coefficient of 2.6 S. It binds F-actin in a Mg2+-dependent, KCl-modulated manner, up to a stoichiometry of about 1 polypeptide per 3.0 actin monomers. In all these properties it is very similar to tropomyosins from higher cells. Antigen-affinity-purified antibodies specifically recognize the Mr 33,000 polypeptide among total yeast proteins and crossreact with bovine brain tropomyosin. In addition, the antibodies specifically crossreact with heat-stable Mr 33,000 polypeptides in extracts of Schizosaccharomyces pombe and Physarum polycephalum. Our detection of tropomyosin in lower eukaryotes suggests that they might have contractile systems very similar to those found in higher organisms.