OBJECTIVE: Mitotane treatment in adrenocortical carcinoma (ACC) results in unreliable measurement of serum total cortisol (TC) levels because of an elevation in corticosteroid-binding globulin (CBG). DESIGN: The use of a newly-developed serum-free cortisol (FC) assay was assessed to investigate the characteristics of a more valid measure of cortisol status. PATIENTS: Sixty-two serum samples from patients with ACC treated with mitotane were studied. Different subgroups were studied according to mitotane levels (20 mg/dl), hydrocortisone replacement treatment, presence of Cushing's syndrome (CS) and adrenocorticotrophin (ACTH) levels. MEASUREMENTS: Serum FC was measured using a newly-developed assay, TC, CBG and plasma ACTH using conventional laboratory kits; TC-to-CBG (Free cortisol index, FCI, nmol/mg) and TC-to-FC (TFR) ratios were calculated. RESULTS: CBG levels were elevated and positively correlated to mitotane levels. FC was positively related to TC and FCI in nearly all subgroups studied. Plasma ACTH was negatively related to parameters of cortisol levels in the total samples studied. In the 'target' subgroup with normal ACTH levels and mitotane levels 14-20 mg/dl, no correlation of plasma ACTH with any parameter studied was seen, and FC suggested over-replacement with hydrocortisone treatment in the subgroup with CS. CONCLUSIONS: FC measurement may offer additional information in the follow-up of patients on mitotane, especially when there is a history of CS which invalidates the use of acute changes in plasma ACTH as a parameter of hydrocortisone replacement. These preliminary data suggest that it may prove useful as a biochemical marker when TC or FCI are invalidated by mitotane treatment or plasma ACTH is suppressed by hypercortisolaemia. Larger studies are needed to substantiate the clinical utility of FC measurement in specific groups of patients.