An improved method for purifying the tryptic fragment (Fragment A) of flagellar ATPase (dynein) from sea urchin spermatozoa is described. The preparation appears homogeneous as judged by ultracentrifugation, electrophoresis on polyacrylamide gels, and immunological techniques. The molecular weight of undenatured Fragment A was determined to be 400,000 and 370,000 by the two methods of disc electrophoresis on polyacrylamide gel and sedimentation equilibrium, respectively. The fragment dissociated into two principal polypeptide chains with molecular weights of 190,000 and 135,000 when heated in the presence of sodium dodecyl sulfate. Antiserum against dynein was prepared in rabbits using purified Fragment A from the sea urchin Anthocidaris crassispina as an antigen. The specificity of this serum toward Fragment A and toward dynein was determined by double diffusion in agarose, by inhibition of ATPase activity, and by sodium dodecyl sulfate-electrophoresis of the antigen-antibody complex. This antiserum also reacted with the enzymes from two other species of sea urchin, Pseudocentrotus depressus and Hemicentrotus pulcherrimus. Analysis of the precipitated antigen-antibody complex showed that the antiserum reacted specifically with the "high molecular weight" polypeptide seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude dynein fractions. This finding supports previous reports that this band derives from dynein ATPase. In our preparations, this "high molecular weight" dynein band appeared single.