Tetracosactin [corticotropin-(1-24)] is used for clinical testing of adrenocortical responsiveness. The usual dose [high dose test (HDT)] is 250 micrograms. With this test, patients with mild secondary adrenal insufficiency are usually not identified, thus putting them at risk of an adrenal crisis in stressful situations. It was recently reported that a tetracosactin test with approximately 1 micrograms [low dose test (LDT)] identifies patients with mild forms of pituitary-adrenal insufficiency. We performed both the HDT and the LDT in 35 control subjects and in 44 patients with pituitary disease, mostly pituitary tumors. In these patients, more sensitive reference tests for evaluating the pituitary-adrenal axis (insulin-induced hypoglycemia, metyrapone, and CRH tests) were also performed. In the HDT, plasma cortisol was measured 30 and 60 min after tetracosactin injection; in the LDT (0.5 microgram/m2 body surface area), plasma cortisol was measured 20, 30, 40, 50, and 60 min postinjection. In 6 control subjects, tetracosactin plasma levels were also measured after injection. In the HDT, the correlation between 30 and 60 min cortisol levels was extremely high (r = 0.991; P < 0.0001), but the correlation of the LDT with the HDT at 30 min was also highly significant (r = 0.948; P < 0.0001). The lower normal limit of cortisol responses (means of controls minus 2 SD) at 30 min was lower in the LDT by 3.1 micrograms/dL (85 nmol/L) than in the HDT. Compared with the reference tests, the diagnostic sensitivities of the HDT and the LDT were almost identical. Both tests identified patients with moderately to severely pathological insulin and metyrapone tests, but not those with slightly pathological reference tests. In the HDT, plasma tetracosactin rose to more than 60,000 pg/mL shortly after injection. In the LDT, it rose to 1,900 pg/mL. Both concentrations stimulate cortisol (supra-) maximally. Together, these data show that in pituitary disorders the results of the LDT and the HDT are almost identical. Plasma tetracosactin levels in the LDT still rise to levels that maximally stimulate the adrenal. Tetracosactin testing with low or high doses cannot generally replace the more expensive and cumbersome insulin or metyrapone tests.