Thermodynamic parameters describing the phage 434 Cro protein have been determined by calorimetry and, independently, by far-UV circular dichroism (CD) measurements of isothermal urea denaturations and thermal denaturations at fixed urea concentrations. These equilibrium unfolding transitions are adequately described by the two-state model. The far-UV CD denaturation data yield average temperature-independent values of 0.99 ± 0.10 kcal mol^-1 M^-1 for m and 0.98 ± 0.05 kcal mol^-1 K^-1 for ΔC_p,U, the heat capacity change accompanying unfolding. Calorimetric data yield a temperature-independent ΔC_p,U of 0.95 ± 0.30 kcal mol^-1 K^-1 or a temperature-dependent value of 1.00 ± 0.10 kcal mol^-1 K^-1 at 25 °C. ΔC_p,U and m determined for 434 Cro are in accord with values predicted using known empirical correlations with structure. The free energy of unfolding is pH-dependent, and the protein is completely unfolded at pH 2.0 and 25 °C as judged by calorimetry or CD. The stability of 434 Cro is lower than those observed for the structurally similar N-terminal domain of the repressor of phage 434 (R1−69) or of phage λ (λ_6-85), but is close to the value reported for the putative monomeric λ Cro. Since a protein's structural stability is important in determining its intracellular stability and turnover, the stability of Cro relative to the repressor could be a key component of the regulatory circuit controlling the levels and, consequently, the functions of the two proteins in vivo.