Dehydroepiandrosterone sulfate (DHEAS), which is of almost exclusive adrenal origin, is important for the androgen status in women and prepubertal children, and DHEAS assays are used in the investigation of hyperandrogenism. There are conflicting reports concerning a diurnal variation in serum DHEAS. Although of adrenocortical origin, serum DHEAS levels are decreased by oral contraceptives (OCs). DHEAS is strongly bound to serum albumin and has a very low metabolic clearance rate. The present study was performed in order to investigate whether a diurnal variation in serum DHEAS exists and, if so, whether this diurnal variation and the decreased DHEAS levels following OC use are related to alterations in adrenocortical steroids or to changes in serum albumin. Serum concentrations of DHEAS, dehydroepiandrosterone (DHEA), cortisol and albumin were determined in blood samples taken every half hour over a 24-h period in 10 healthy women before and during use of combined OCs. Significant and frequently synchronous diurnal variations in serum DHEAS and albumin were found before as well as during OC use. These variations were not synchronous with the diurnal variation in DHEA. OCs significantly decreased serum DHEAS and albumin levels. A multiple regression analysis showed changes in albumin to be the most decisive factor for the diurnal variation as and for OC-induced changes in DHEAS. Changes in serum DHEAS during the day and following OC use are related to alterations in its main binding protein, serum albumin, rather than to changes in adrenocortical steroid secretion.