Urinary corticosteroid excretion patterns with special emphasis on the excretion of cortisol and 2α-hydroxycortisol were determined in individual short-haired English type guinea pigs. Strain 13 and strain 13 × Hartley F1 hybrid guinea pigs exhibited a significant daily variability in excretion above that due to the method alone. There was a significant diurnal variation in the excretion of 2α-hydroxy cortisol and cortisol with higher levels during the day in most animals. The ratio of cortisol: 2α-hydroxy cortisol during the day and night was not significantly different. Following ACTH administration no prolonged period of low corticosteroid excretion was found after the high excretion subsided. There was no consistently significant correlation between urine volume and the excretion of 2α-hydroxycortisol and cortisol in the majority of the animals. Immobilization caused an approximately 2- to 3-fold increase in corticosteroid excretion, which was only about half that found following ACTH, but with no significant change in the ratio of cortisol: 2α-hydroxycortisol. Fatal illness of probably infectious origin caused characteristic changes in the excretion patterns, with an increased excretion of 2α-hydroxycortisol and a decreased excretion of cortisol sometime before death in the strains excreting high 2α-hydroxycortisol. Pitressin caused a 2- to 3-fold increase in the excretion of 2α-hydroxycortisol and cortisol in strain 13 guinea pigs. (Endocrinology75: 226, 1964)