Microtubules were isolated from unfertilized eggs of the sea urchin with the use of the anti-tumor drug taxol. In addition to tubulin, prominent high molecular weight (Mr 205,000-350,000) microtubule-associated proteins (MAPs) were identified as well as MAP species of Mrs 77,000, 100,000, and 120,000. The microtubules were covered with both short periodic arms and longer filamentous arms, both classes of which appeared to crosslink the microtubules into bundles. Monoclonal antibodies were prepared to an unfractionated MAPs preparation. We isolated clonal hybridoma lines producing antibodies to tubulin and to four non-tubulin proteins of Mrs 235,000, 205,000, 150,000, and 37,000. All antibodies strongly and specifically stained the mitotic spindle of dividing sea urchin eggs. All four of the immunoreactive, non-tubulin species behaved as MAPs during microtubule isolation. Thus, we have identified a variety of sea urchin MAPs by biochemical, ultrastructural, and immunochemical means. The immunochemical experiments demonstrated that four of these proteins are microtubule-associated components of the mitotic spindle. We suggest that those proteins that we observed as cross-bridges between the isolated microtublules may be either structural or functional components of the spindle.