We have generated a series of monoclonal antibodies against axonemal proteins from sea urchin spermatozoa in order to identify novel proteins involved in the regulation of flagellar motility. The monoclonal antibody D405-14 inhibited the motility of demembranated-reactivated sperm models at low concentrations and recognized a single polypeptide of 33 kDa (p33) on immunoblots of sea urchin axonemal proteins. Fractionation of the axonemes with high salt solutions, heat, and detergent resulted in the selective extraction of p33 into a 0.6 M NaCl-soluble and a 0.5% sodium lauryl sarcosinate (Sarkosyl)-soluble form. Both forms of p33 were purified to apparent homogeneity by immunoaffinity chromatography on monoclonal antibody D405-14-Sepharose. We have also isolated and sequenced a full-length cDNA clone encoding the 33-kDa protein. The sequence predicts a polypeptide of 260 amino acids having a mass of 29,730 Da and an isoelectric point of 9.3. Sequence comparison indicates that p33 is 66% identical (74% similar) to the p28 light chain of axonemal inner dynein arm of Chlamydomonas reinhardtii. Taken together, these results suggest that we have identified a p28 light chain homolog in sea urchin sperm axoneme and that this protein may play a dynamic role in flagellar motility.