The folding of recombinant hepatitis B virus X-protein (rHBx) solubilized from Escherichia coli inclusion bodies was investigated. By sequential dialysis of urea, rHBx was folded into its native structure, which was demonstrated by the efficacy of its transcriptional activation of the adenovirus major late promoter (MLP), fluorescence spectroscopy, and circular dichroism (CD) analysis. The decrease in CD values at 220 nm and a corresponding blue shift of the intrinsic fluorescence emission confirmed the ability of rHBx to refold in lower concentrations of urea, yielding the active protein. Equilibrium and kinetic studies of the refolding of rHBx were carried out by tryptophan fluorescence measurements. From the biphasic nature of the fluorescence curves, the existence of stable intermediate states in the renaturation process was inferred. Reverse phase-high performance liquid chromatography (RP-HPLC) analysis further demonstrated the existence of these intermediates and their apparent compactness.