Pepsin exists as alkaline denatured state (I-p) in pH range 8-10, where the N-terminal domain of the protein is mostly unfolded while the C-terminal domain is intact. The effects Of fluorinated (TFE) and non-fluorinated (methanol) organic solvents on this partially unfolded state (I-p) of pepsin were investigated using various spectroscopic methods. Both, fluorinated (TFE) and non-fluorinated (methanol) organic solvents induce secondary structure (a-helix) after a critical concentration. The I-p state of pepsin unfolds in cooperative manner but the transition was found to be non-cooperative in the presence of 40% methanol or TFE. The differences in the unfolding of the protein in the presence and the absence of these organic solvents were interpreted. Our results indicate that unfolding transitions in I-p state are mostly dominated by unfolding of C-terminal domain because the N-terminal domain is largely unstructured in this state. The organic solvents (TFE and methanol) induce more secondary structure in N-terminal domain and make it another unfolding entity with different stability compare to C-terminal resulting into sequential unfolding of the domain. (C) 2009 Elsevier Ltd. All rights reserved.