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 (J1→b), transepithelial voltage (VT), and water permeability (Pf) in CCD from deoxycorticosterone (DOC)-treated rats. lonomycin (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 Jl→b or Pf, 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 Jl→b or Pf. OAG at 50 μM had no effect on VT or transepithelial resistance, indicating no inhibition of conductive Na+ transport. We conclude that increased [Ca2+]i and PKC activation do not affect Jl→b or Pf in the rat CCD. These findings may account for the sustained increase in Jl→b produced in the rat CCD by AVP. Copyright © 1993 the American Physiological Society.