The rates of CO binding to cytochrome oxidase at low temperatures were studied in mitochondria isolated from the hearts of 3- and 30-32-month-old male F344/N rats. A single apparent energy of activation of 9.6 kcal/mol is observed in mitochondria from 3-month-old rats in the presence of 1% CO. In the presence of 100% CO, the energy of activation is 10.1 kcal/mol and the rate constants of CO recombination following flash photolysis are approximately twice the rate constants in the presence of 1% CO at warm temperatures indicating that an intermediate region near the heme iron can hold a maximum of two CO molecules. In 30-month-old cardiac mitochondria, recombination in the presence of 1% and 100% CO requires crossing barriers of 9.4 kcal/mol and 10.3 kcal/mol height, respectively. The approximate doubling of the values of k at warm temperatures (above 225 K) indicates, as in mitochondria from young animals, that CO migration from solvent to the heme iron involves migration across two similarly sized barriers separating the Fe from an innermost intermediate region I (capable of holding only one CO) and separating region I from intermediate region I2 (capable of holding two CO). The kinetics of CO binding to cytochrome oxidase do not change with increasing age.