The short-term regulation of multidrug resistance-associated protein 3 (Mrp3/MRP3) by cAMP and protein kinase C (PKC) was investigated in sandwich-cultured rat and human hepatocytes and isolated perfused rat livers. The modulators glucagon (500 nM) and the phorbol ester, PMA (0.1 µM), were utilized to increase intracellular cAMP and PKC levels, respectively. In glucagon-treated rat hepatocytes, efflux of the Mrp3 substrate 5-(6)-carboxy- 2',7'dichlorofluorescein (CDF) increased ~1.5 fold, and in the presence of the Oatp inhibitor sulfobromophthalein (BSP), the same increase in CDF efflux was observed relative to BSP treatment alone. Confocal microscopy revealed more concentrated Mrp3 fluorescence in the basolateral membrane (less diffuse staining pattern) with glucagon treatment. PMA had no effect on either Mrp3 activity or localization in sandwich-cultured rat hepatocytes. Glucagon and PMA treatment in isolated perfused rat livers resulted in a 3-fold increase (14 ± 4.6 µl/min/g liver) and 4-fold decrease (1.3 ± 0.3 µl/min/g liver) in CDF basolateral clearance, respectively, compared to control livers (4.7 ± 2.3 µl/min/g liver), while CDF biliary clearance was not statistically different. In sandwich-cultured human hepatocytes, glucagon treatment resulted in a 1.3-fold increase in CDF efflux and a concomitant increase in MRP3 fluorescence in the basolateral membrane. In summary, cAMP and PKC appear to be involved in the short-term regulation of Mrp3/MRP3 as demonstrated by alterations in both activity and localization in rat and human hepatocytes.