The studies of GroEL, almost exclusively, have been concerned with the function of the chaperonin under non-stress conditions, and little is known about the role of GroEL during heat shock. Being a heat shock protein, GroEL deserves to be studied under heat shock temperature. As a model for heat shock in vitro, we have investigated the interaction of GroEL with the enzyme rhodanese undergoing thermal unfolding at 43 degrees C. GroEL interacted strongly with the unfolding enzyme forming a binary complex. Active rhodanese (82%) could be recovered by releasing the enzyme from GroEL after the addition of several components, e.g. ATP and the co-chaperonin GroES. After evaluating the stability of the GroEL-rhodanese complex, as a function of the percentage of active rhodanese that could be released from GroEL with time, we found that the complex had a half-life of only one and half-hours at 43 degrees C; while, it remained stable at 25 degrees C for more than 2 weeks. Interestingly, the GroEL-rhodanese complex remained intact and only 13% of its ATPase activity was lost during its incubation at 43 degrees C. Further, rhodanese underwent a conformational change over time while it was bound to GroEL at 43 degrees C. Overall, our results indicated that the inability to recover active enzyme at 43 degrees C from the GroEL-rhodanese complex was not due to the disruption of the complex or aggregation of rhodanese, but rather to the partial loss of its ATPase activity and/or to the inability of rhodanese to be released from GroEL due to a conformational change.