The initiation of the DNA polymerase III holoenzyme-catalyzed reaction is blocked by antibody directed against the beta subunit; elongation is unaffected (Johanson, K., and McHenry, C. (1980) J. Biol. Chem. 255, 10984-10990). We have developed an immunological method for quantitating nanogram quantities of beta in reaction complexes. Using this method, we have demonstrated that beta is present in all stages of the DNA polymerase III holoenzyme reaction. Upon initiation complex formation, the antigenic determinants of beta become inaccessible to anti-beta immunoglobulin G. The methods described herein should be generally applicable to the study of a variety of multienzyme complexes. Even after conversion of a primed G4 single strand to the duplex replicative form, beta does not readily dissociate. This creates a kinetic barrier to the overall holoenzyme replicative reaction.