Autoclaving diminishes the nutritional value of rat diets, depending on the duration and temperature of the process and the type of dietary protein. We evaluated in vivo and in vitro the effects of autoclaving on the protein and energy values of soy-free and soy-containing rat diets. The true digestibility and biological value of the dietary protein were determined in a 10-d experiment involving 28-d-old Wistar Crl:WI(Han) male rats fed casein- or soy-containing diet that was autoclaved for 20 min at 121 °C (T1), 10 min at 134 °C (T2), or not autoclaved (T0). The apparent protein digestibility and metabolizable energy concentration of experimental diets were assayed during an 18-d trial involving 6-wk-old Wistar-Crl:WI(Han) male rats and compared with a commercial diet. The neutral detergent fiber (NDF) content, amount of protein bound to NDF, protein solubility, and in vitro ileal protein digestibility were determined. Autoclaving decreased protein solubility, with the T2 condition having a greater effect than that of T1, and decreased the protein parameters determined in vivo, except for the apparent digestibility of the standard rat diet. Autoclaving decreased metabolizable energy slightly. The Atwater formula yielded higher values than those determined in rats, in vitro, and calculated according to the pig equation. We conclude that autoclaving diets according to the T1 program was less detrimental to dietary protein than was T2 and that the NDF content and protein solubility may be helpful in assessing the effect of autoclaving. The pig formula and in vitro method appear to be valid for estimating the metabolizable energy of rat diets.