The ClpAP protease from Escherichia coli consists of the ATP-binding regulatory component, ClpA (subunit Mr 84 165), and the proteolytic component, ClpP (subunit Mr 21 563). Our hydrodynamic studies demonstrate that the predominant forms of these proteins in solution correspond to those observed by electron microscopy. ClpP and proClpP(SA), which in electron micrographs appear to have subunits arranged in rings of seven subunits, were found by ultracentrifugation to have s20,w values of 12.2 and 13.2 S and molecular weights of 300 000 and 324 000 +/- 3000, respectively, indicating that the native form of each consists of two such rings. The two intact rings of ClpP were separated in the presence of >/= 0.1 M sulfate at low temperatures, suggesting that ring-ring contacts are polar in nature and more easily disrupted than subunit contacts within individual rings. Sedimentation equilibrium analysis indicated that ClpA purified without nucleotide exists as an equilibrium mixture of monomers and dimers with Ka = (1.0 +/- 0.2) x 10(5) M-1 and that, upon addition of MgATP or adenosine 5'-O-(3-thiotriphosphate), ClpA subunits associated to a form with Mr 505 000 +/- 5000, consistent with the hexameric structure seen by electron microscopy. Sedimentation velocity and gel-filtration analysis showed that the nucleotide-promoted hexamer of ClpA (s20,w = 17.2 S) binds tightly to ClpP producing species with s20,w values of 21 and 27 S (f/f0 = 1.5 and 1.8, respectively), consistent with electron micrographs of ClpAP that show a single tetradecamer of ClpP associated with either one or two ClpA hexamers [Kessel et al. (1995) J. Mol. Biol. 250, 587-594]. Under assay conditions in the presence of ATP and Mg2+, the apparent dissociation constant of hexameric ClpA and tetradecameric ClpP was approximately 4 +/- 2 nM. By the method of continuous variation, the optimal ratio of ClpA to ClpP in the active complex was 2:1. The specific activities of limiting ClpA and ClpP determined in the presence of an excess of the other component indicated that the second molecule of ClpA provides very little additional activation of ClpP.