The effects of monoclonal anti-tubulin antibodies on the motility of demembranated and reactivated sea urchin spermatozoa were investigated. Two out of ten antibodies examined significantly reduced the motility of spermatozoa, both in motile rate and swimming speed. The binding patterns of the two antibodies YL1/2 and TUB2.1 to the axoneme were studied by immunoblot, immunofluorescence, and immunoelectron microscopy. YL1/2 bound to the axoneme in a specific pattern; signals were very intense in the tail, rich in the proximal portion, and scarce in the middle part of the axoneme. Because the inhibitory effects of the antibody on the motility of spermatozoa with fully long flagella and short flagella were similar, the inhibition was probably due to the binding of the antibody to the proximal portion of the flagellum. TUB2.1 evenly bound to the axoneme by immunofluorescence and immunoelectron microscopy. On the other hand, the eight antibodies which did not affect sperm motility, did not bind to unfixed axonemes, although epitopes for these antibodies were detected abundantly in the axoneme.