Escherichia coli trp aporepressor (TR) is a highly helical, dimeric protein whose folding has been shown to involve three phases whose relaxation times range from 200 ms to 50 s at 25 degrees C and pH 7.6 [Gittelman, M. S., & Matthews, C. R. (1990) Biochemistry 29, 7011-7021]. All three phases are urea and protein concentration independent below 3 M urea, suggesting that cis/trans proline isomerization might limit the folding of TR under these conditions. This hypothesis was tested by measuring the sensitivity of the folding reaction to site-directed mutagenesis and to cyclophilin, a peptidyl-prolyl isomerase. Each of the four proline residues in TR was replaced singly as well as simultaneously, and the effects on the folding mechanism were assessed. All of these mutants, including the version lacking prolines (des-Pro TR), retain three slow, denaturant-independent folding phases similar to those observed for wild-type TR. However, the pattern of catalysis of the two slower folding phases in wild-type and mutant TRs by cyclophilin shows that cis/trans isomerization of the Thr44/Pro45 peptide bond can limit folding in proteins containing Pro45. The observation of three urea-independent, slow folding phases in des-Pro TR demonstrates that proline isomerization is not solely responsible for this complex folding behavior. Other types of isomerization or conformational rearrangement reactions appear to limit the folding of this dimeric protein under strongly folding conditions.