To obtain insight regarding the mechanism(s) of response of the catalytic unit of glucose-6-phosphatase (D-glucose-6-P phosphohydrolase; EC 3.1.3.9) to glucocorticoid administration and insulin deprivation, the functional enzyme concentration E0 was estimated from presteady-state kinetics by the stopped-flow technique. The E0 values were compared with Vmax values determined by the steady-state kinetic approach. Studies were carried out with detergent-disrupted microsomes from livers of normal fed, 48-h fasted, streptozotocin-diabetic, and triamcinolone-treated rats. All of the treatments caused an increase in E0, but Vmax values were increased only in fasting and diabetes. Km values were unaffected by all the treatments. The increase in Vmax observed with fasted and diabetic rats was explained by an increase in E0 alone. These results showed that insulin deprivation resulted in an increased formation of fully active glucose-6-phosphatase catalytic unit. In contrast, administration of triamcinolone caused an increase in E0 but not in Vmax. It was concluded that glucocorticoid administration may promote formation of catalytic units of glucose-6-phosphatase which are less active than the enzyme normally present or formed in response to insulin deprivation.