The double ring chaperonin GroEL binds unfolded protein, ATP, and GroES to the same ring, generating the cis ternary complex in which folding occurs within the cavity capped by GroES (cis folding). The functional role of ATP, however, remains unclear since several reports have indicated that ADP and AMPPNP (5'-adenylyl-beta,gamma-imidodiphosphate) are also able to support the formation of the cis ternary complex and the cis folding. To minimize the effect of contaminated ATP and adenylate kinase, we have included hexokinase plus glucose in the reaction mixtures and obtained new results. In ADP and AMPPNP, GroES bound quickly to GroEL but bound very slowly to the GroEL loaded with unfolded rhodanese or malate dehydrogenase. ADP was unable to support the formation of cis ternary complex and cis folding. AMPPNP supported cis folding of malate dehydrogenase to some extent but not cis folding of rhodanese. In the absence of hexokinase, apparent cis folding of rhodanese and malate dehydrogenase was observed in ADP and AMPPNP. Thus, the exclusive role of ATP in generation of the cis ternary complex is now evident.