The 5-subunit form of the Escherichia coli F1-ATPase, characterized by the subunit composition alpha 3 beta 3 gamma delta epsilon, failed to exhibit a rate acceleration when samples of the enzyme hydrolyzing substoichiometric concentrations of [gamma-32P]ATP were switched from unisite to multisite hydrolysis by the addition of a cold chase. A 4-subunit form of the enzyme lacking in the delta subunit (alpha 3 beta 3 gamma epsilon) did exhibit cold chase-promoted accelerations in the hydrolysis of ATP. Reconstitution of a 5-subunit enzyme by incubating the 4-subunit form of the enzyme with a purified preparation of subunit delta was accompanied by a disappearance in the response to a cold chase. The rate constants and equilibrium constants for unisite catalysis by the 4-subunit enzyme did not differ significantly from previously reported values that may have been based on a mixture of 4- and 5-subunit forms of the enzyme. The vesicular form of Escherichia coli F0F1-ATPase exhibited a response to a cold chase only if the vesicles were first extracted with KCl. [gamma-32P]ATP bound in the high affinity catalytic sites of KCl-extracted membranes partially dissociated in an energy-dependent manner when the vesicles oxidized NADH.