Circular dichroism was used to monitor the thermal unfolding of ribonuclease A in 50% aqueous methanol. The spectrum of the protein at temperatures below −10°C (pH* 3.0) was essentially identical to that of native ribonuclease A in aqueous solution. The spectrum of the thermally denatured material above 70°C revealed some residual secondary structure in comparison to protein unfolded by 5 M Gdn·HCl at 70°C in the presence or absence of methanol. The spectra as a function of temperature were deconvoluted to determine the contributions of different types of secondary structure. The position of the thermal unfolding transition as monitored by α-helix, with a midpoint at 38°C, was at a much higher temperature than that monitored by β-sheet, 26°C, which also corresponded to that observed by ΔA286, tyrosine fluorescence and hydrodynamic radius (from light scattering measurements). Thus, the loss of β-sheet structure is decoupled from that of α-helix, suggesting a step-wise unfolding of the protein. The transition observed for loss of α-helix coincides with the previously measured transition for His-12 by NMR from a partially folded state to the unfolded state, suggesting that the unfolding of the N-terminal helix in RNase A is lost after unfolding of the core β-sheet during thermal denaturation. The thermally denatured protein was relatively compact, as measured by dynamic light scattering.