Physiologically as well as industrially, alpha-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer alpha-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both alpha and beta structures in Cicer alpha-galactosidase and showed that its secondary structure belongs to alpha + beta class of proteins with 31 % alpha-helicity. For Cicer alpha-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH 2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH 2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer alpha-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40 A degrees C; however, above this temperature, denaturation was observed.