Previous studies have demonstrated that glucocorticoids alter biotin metabolism. To extend these studies, the effect of dexamethasone on biotin pools was analyzed in rats consuming a purified diet containing a more physiological level of dietary biotin intake (0.06 mg/kg). Acute (5 h) dexamethasone administration (0.5 mg/kg) elicited elevated urinary glucose output as well as elevated urinary biotin excretion and serum biotin. Renal and hepatic free biotin was also significantly elevated by acute dexamethasone administration. Chow-fed rats treated with an acute administration of dexamethasone demonstrated significantly elevated urinary glucose excretion, urinary biotin excretion, and serum biotin, but no change in tissue associated biotin was detected. Chronic administration of dexamethasone (0.5 mg/kg ip) over 4 days significantly elevated urinary glucose excretion 42% but had no effect on urinary biotin excretion, serum biotin, or hepatic- or renal-associated free biotin. These results demonstrate the existence of potentially novel regulatory pathways for total biotin pools and the possibility that experimental models with high initial biotin status may mask potentially important regulatory mechanisms.