AbstractIn current purification strategies, affinity for microtubules or calmodulin is used to identify and purify cytoplasmic dynein‐like ATPase from cell‐free extracts of unfertilized sea urchin eggs. However, affinity purification procedures, though they define dynein‐like ATPase activity, have not yet proven to be quantitative. An alternative purification strategy capable of producing a high yield of enzyme would require a specific assay in order to monitor cytoplasmic dynein purity at each step.In this study, we make a detailed comparison of the effects of EHNA on 22 different ATP‐metabolizing enzyme activities, including 13 Mg++‐ATPases. We isolate cytoplasmic dynein‐like ATPase activity from three species of sea urchin eggs and sperm and show by means of dose‐response curves that their sensitivities to inhibition by EHNA are very similar to one another. We demonstrate further that the EHNA dose‐response characteristics of fourteen other ATP‐metabolizing enzyme activities, including seven nondynein Mg++‐ATPases, differ quantitatively from those of dynein‐like ATPases.In studies of three other agents (vanadate, Ca++/calmodulin, and Triton X‐100), we find that dynein‐like ATPases vary by two orders of magnitude in their sensitivities to inhibition by vanadate, and little or no stimulation by either Ca++/calmodulin or Triton X‐100 is seen. Our results suggest that inhibition by EHNA is a universal and specific property of dynein‐like ATPases, which ultimately should prove useful in the quantitative purification and characterization of cytop