The thermodynamic properties and phase diagram of the thermal-induced fibrous aggregation of cytochrome c with a simple organic co-solvent, 1,4-dioxane, were determined by differential scanning calorimetry, circular dichroism, and scanning electron microscopy at various concentrations of 1,4-dioxane. At x = 0.2-0.3 mol fraction of 1,4-dioxane, cytochrome c transformed to an alpha helix-rich denatured state at 25 degrees C, which transformed into a fibrous aggregation upon heating. The enthalpy change for the fibrous aggregation, -456 kJ/mol = 0.30), was numerically greater than the thermal denaturation of the native state, 298 kJ/mol (x = 0), indicating that aggregate formation involved a conformation with many intermolecular interactions. At x > 0.3, cytochrome c aggregated and precipitated at 25 degrees C without heating, and the precipitate transformed into an amorphous state. These results suggest that the previous state is important for transition into the fibrous state. Thermodynamic data on the use of co-solvents for globular proteins will facilitate understanding of the aggregation mechanism.