Microtubules assembled from sea urchin eggs with the use of taxol contain a 77,000-dalton protein as the major nontubulin component [Vallee and Bloom (1983): Proc Natl. Acad. Sci. U.S.A. 80:6259-6263]. We have raised five monoclonal antibodies to this protein to aid in its characterization. Immunoblot analysis of the sea urchin microtubule purification fractions indicated that the protein copurified quantitatively with microtubules. All five antibodies stained the mitotic spindle of dividing sea urchin eggs by immunofluorescence microscopy, indicating that the protein was a component of the mitotic spindle and suggesting that it was actually localized on microtubules in vivo. Immunofluorescent staining of higher resolution was observed in a subpopulation of the coelomic cells found in adult sea urchins, confirming that the 77,000-dalton protein is indeed present on microtubules in vivo. Because taxol was not used for the immunofluorescence experiments, we conclude that the microtubule-associated protein (MAP)-like behavior of the 77,000-dalton protein in vitro was not induced artifactually by taxol. To determine whether this protein is a component of sea urchin microtubules in general, cilia obtained from blastula stage embryos and sperm tail flagella were analyzed with the antibodies. The protein was undetectable by both immunoblot analysis and immunofluorescence microscopy in both preparations of axonemal microtubules. These results indicated that the 77,000-dalton MAP is restricted to cytoplasmic and mitotic microtubules in the sea urchin. Furthermore, in view of its particular abundance in embryos, whose microtubules are devoted substantially to mitosis, the 77,000-dalton MAP is likely to play an important role in regulating the activity of mitotic spindle microtubules in the sea urchin.