It is well established that alpha-cyclodextrin (alpha-CD) is an effective refolding aid for some proteins in dilution additive mode. On the other hand, it has recently been shown that guanidine hydrochloride (GuHCl), the well known protein denaturant, acts, at well defined mild concentrational ranges, as a stabilizer of the "molten globule" intermediates with the final renaturation as well as aggregation of the denatured protein under investigation. In the present study, the effect of GuHCl on alpha-CD efficacy on protein renaturation was investigated. The unassisted and alpha-CD-assisted refolding of chemically denatured alpha-amylase in the presence of various alpha-CD and GuHCI concentrations were evaluated, using activity assay, far-UV circular dichroism (CD) spectroscopy, turbidimetric analysis and hydrophobic probe 8-anilinonaphtalene-1-sulfonic acid (ANS) fluorescence techniques. The recovered activity, average helical structure and the extent of dilution-induced aggregation of refolded alpha-amylase were affected with increase of final GuHCI concentration in dilution buffer, especially in alpha-CD-assisted refolded samples. The refolding yield and protein helicity decreased, while apparent turbidity due to protein aggregation (A(lim)) and its rate were increased. The results clearly indicated that alpha-cyclodextrin effectively suppressed aggregation, especially at lower GuHCl concentrations in the refolding buffer. Based on these results, application of low dilution factors in alpha-CD-dependent protein renaturation systems should be avoided due to inhibitory effects of high final denaturant concentration on alpha-CD efficacy besides its direct influence on the production of aggregation-prone intermediate(s) of protein molecules. (c) 2005 Elsevier Ltd. All rights reserved.