Experiments examined the effects of elevation of intracellular calcium concentration ([Ca2+](i)) or activation of protein kinase C (PKC) on Na+ and water transport in the rat cortical collecting duct (CCD). We measured the lumen-to-bath 22Na+ flux (J(l→b)), transepithelial voltage (V(T)), and water permeability (P(f)) in CCD from deoxycorticosterone (DOC)-treated rats. Ionomycin (0.5 and 1 μM) and thapsigargin (1 and 2 μM) were used to increase [Ca2+](i). Phorbol 12-myristate 13-acetate (PMA; 0.3 and 1 μM) and oleoyl-acetyl-glycerol (OAG; 100 μM) were used as activators of PKC. [Ca2+](i) was measured in isolated perfused tubules using the fluorescent dye fura 2. When added to the bathing solution, 220 pM arginine vasopressin (AVP) failed to affect [Ca2+](i), whereas 1 μM ionomycin increased [Ca2+](i) by 103 ± 15% and 2 μM thapsigargin increased [Ca2+](i) by 24 ± 4%. In flux studies, neither ionomycin nor thapsigargin affected J(l→b) or P(f), although ionomycin caused marked morphological changes. Ionomycin also failed to alter either parameter in tubules from non-DOC-treated rats. Neither 100 μM OAG nor 1 μM PMA affected J(l→b) or P(f). OAG at 50 μM had no effect on V(T) or transepithelial resistance, indicating no inhibition of conductive Na+ transport. We conclude that increased [Ca2+](i) and PKC activation do not affect J(l→b) or P(f) in the rat CCD. These findings may account for the sustained increase in J(l→b) produced in the rat CCD by AVP.