The influence of short-chain alcohols and osmolytes on the structural properties, catalytic activity and thermal stability of myoglobin (Mb) has been investigated. Results show that in the presence of a cosolvent, the catalytic activity of Mb decrease as the concentration increase, starting when the concentration was low. Spectroscopic investigations show that changes in catalytic activity are not due to protein conformational changes. Otherwise, no significant correlation between the thermal stability and the catalytic activity is evident from the data. Our measurements show a correlation between the effect of cosolvents on catalytic activity and changes in the microstructure of solvent, as inferred from previous IR data on these mixtures. (C) 2009 Published by Elsevier B.V.