The refolding of barnase in the presence of GroEL has been monitored on the millisecond to seconds time scale using stopped-flow kinetics. GroEL binds rapidly and tightly to the denatured enzyme with a second-order rate constant of greater than 1.3 x 10(8) s-1 M-1 and slows down greatly the rate of barnase refolding. However, addition of ever increasing concentrations of GroEL does not prevent barnase refolding completely, as would be expected from mass action if folding of barnase could proceed only in free solution. At saturating concentrations of GroEL, barnase refolds with a half-life of 30 s, compared with 50 ms for refolding of free enzyme. The rate-determining step in the refolding of free barnase is the reaction of a "late" folding intermediate. A mutant of barnase that fold more slowly (Ser-->Ala91), refolds at a correspondingly lower rate when bound to GroEL, suggesting that formation of the fully folded state may be rate limiting for folding on GroEL. For the slow-folding Ser-->Ala91 mutant, the rate-determining refolding step has a half-life of 180 ms. In sequential mixing experiments, a delay was introduced to allow the Ser-->Ala91 mutant to refold for 30 ms before being mixed with GroEL. This reduces by 50% the amount of mutant barnase initially bound by GroEL. As only 11% of this mutant barnase is fully refolded from the late intermediate in 30 ms, there is preferential binding of an earlier refolding state to GroEL. We show by single mixing experiments that binding, not hydrolysis, of ATP reduces the lag in regain of barnase activity seen with GroEL alone. In the presence of high concentrations of ATP and GroEL the rate constant for refolding of barnase approaches that found in their absence, probably because ATP reduces the affinity of GroEL for refolding barnase, such that bound barnase is released and refolds unhindered. The addition of exceedingly small quantities of GroES in the presence of excess GroEL and a moderate amount of ATP also has a marked effect on the barnase refolding rate constant, suggesting that GroES may have higher affinity for the barnase: GroEL complex than for GroEL.