In patients with Cushing's disease (CD), basal inferior petrosal sinus arginine vasopressin (AVP) concentrations are greater than peripheral levels and are further increased by the administration of CRH. AVP has an interpetrosal sinus gradient similar to that for ACTH, leading to the hypotheses that petrosal sinus AVP might either be derived from the corticotroph adenoma or be important for adenoma formation. To determine whether petrosal sinus AVP is truly increased in patients with CD, we compared inferior petrosal sinus and peripheral venous AVP and ACTH levels in 23 patients with CD and 9 healthy volunteers before and after iv ovine CRH. In both groups, AVP and ACTH showed interpetrosal lateralization, such that greater levels of both hormones were found at each time point in a single dominant petrosal sinus. When both hormones exhibited lateralization (an intersinus gradient > 1.5), ACTH and AVP always lateralized together. In patients with CD, the ACTH interpetrosal sinus lateralization correctly identified the side of the pituitary containing the tumor in 75% of evaluable patients, whereas the AVP interpetrosal sinus lateralization identified 63% (P = NS). Ovine CRH stimulated AVP in both the dominant and nondominant petrosal sinuses in patients with CD. Although basal AVP in the dominant petrosal sinus was not significantly different in patients with CD and normal volunteers (144 +/- 85 vs. 13.0 +/- 4.3 pmol/L; P = 0.058), dominant petrosal sinus AVP was significantly elevated in patients with CD compared to normal volunteers at 3 min (269 +/- 122 vs. 45.1 +/- 30.0 pmol/L; P < 0.05) and 5 min (315 +/- 120 vs 40.2 +/- 23.6 pmol/L; P < 0.05) after ovine CRH administration. Peripheral venous AVP levels were similar in all groups. We conclude that lateralization of AVP secretion occurs in both patients with CD and normal volunteers, but there is greater CRH-stimulated AVP secretion in the inferior petrosal sinuses of patients with CD.