Chromobindin A is a large, multisubunit protein that binds to chromaffin granule membranes in a Ca2+- and ATP-regulated manner. Ca2+ stimulates binding to the membrane, whereas ATP, in the the absence of Ca2+, is required for release of the protein from the membrane. We now report that spectral and HPLC data indicate that nucleotides are associated with the native chromobindin A complex and that the protein can bind two molecules of [3H]ATP in vitro. Chromobindin A also appears to be a novel nucleotide triphosphatase. ATPase activity was detected in fractions containing chromobindin A isolated by affinity chromatography, gel filtration, or ion exchange chromatography. Kinetic studies indicated that the Vmax is 44 nmol of Pi/mg/min and the Km is 0.115 mM, whereas the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate acts as a competitive inhibitor of this reaction with a Ki of 0.08 mM. The activity was found to be sensitive to protease treatment or to preincubation at 65 degrees C and was inhibited by Ca2+ or low pH. The ATPase activity was not inhibited by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, vanadate, oligomycin, or azide.