The contractile properties of the myonemes of Stentor are very similar to caltractin (centrin)-containing fibers of other organisms. We investigated whether the calcium-binding protein caltractin was present in Stentor by using three different antibodies to caltractin or caltractin-related proteins, in conjunction with immunofluorescence microscopy and protein blotting. Immunofluorescence demonstrated that a protein immunologically similar to caltractin is present in the myonemes and in the bases of the membranelles of Stentor. The localization to the myonemes is observed in intact cells, osmotically lysed cells, and isolated cortices. Double-label immunofluorescence with anti-alpha-tubulin and anti-caltractin antibodies showed that the fluorescence in the myonemes was not in the overlying Km fibers. The myonemes in the posterior one-third of the cell appear as thick fibers with no cross-bridging. They become thinner as they approach the anterior end of the cell and show extensive cross-bridging here. Staining in the bases of the membranelles shows a distinct comma-like immunofluorescence pattern similar to that seen with protargol-stained cells and SEM views of the membranellar band reported by others. Western blots demonstrated that the caltractin-like protein in Stentor has an apparent molecular weight of 23 kDa compared with the 20-kDa protein from Chlamydomonas and is a calcium-binding protein.