The adrenal gland uptake of 131I-6-beta-iodomethyl-19-norcholesterol (NP-59) was calculated using a semioperator-independent computer algorithm in 17 patients with Cushing's syndrome (CS). Twelve had ACTH-dependent and 5 had ACTH-independent CS. The mean adrenal gland uptake (percentage of administered dose) of NP-59 was 0.74 +/- 0.18% (range, 0.21-2.02%) in CS and was significantly higher than that of normal subjects (0.33 +/- 0.02%). Patients with the ectopic ACTH syndrome and ACTH-independent CS manifested the highest uptakes (mean, 1.18 +/- 0.08%; range, 0.74-2.02%). In the patients with ACTH-dependent CS, a significant correlation was observed between 24-h urinary free cortisol excretion and NP-59 adrenal gland uptake (r = 0.97, P less than 0.001). No relationship was seen between NP-59 uptake and urinary free cortisol in ACTH-independent CS or between NP-59 uptake and urinary 17-hydroxycorticosteroids, 17-ketosteroids, cortisol secretion rate, plasma cortisol, or ACTH levels in either ACTH-dependent or ACTH-independent CS. We conclude that in addition to localizing the sites(s) of adrenocortical hypersecretion in CS, the level of NP-59 adrenal uptake is a reflection of cortisol excretion in ACTH-dependent disease and may be utilized as another parameter to quantitate adrenal disease activity in CS.