1. Effects of prostaglandin E2 (PGE2) on ion transport were examined by observing the transmural (VT) and basolateral membrane voltage (VB) in the in vitro perfused rabbit connecting tubule (CNT) and the cortical collecting duct (CCD). 2. Addition of 1 microM PGE2 to the bath induced a biphasic response of transmural voltage (VT), with initial negative VT deflection followed by positive deflection in the CNT, but monophasic negative deflection in the CCD. Because PGE2 had no affect on the basolateral membrane voltage (VB), PGE2 mainly causes changes in the apical membrane voltage. 3. Elimination of Na+ from the lumen abolished the PGE2-induced VT response in the CNT. In the presence of 10 microM luminal amiloride, PGE2 caused only an initial negative deflection without causing later positive deflection. The positive VT deflection induced by PGE2 in the CCD was also blocked by luminal amiloride. 4. Addition of ouabain (0.1 mM) to the bath completely abolished the PGE2-induced VT changes in the CNT, indicating that an intact Na(+)-K+ pump is a prerequisite for the VT response to PGE2. 5. Addition of 2 mM Ba2+ to the lumen did not affect biphasic VT response to PGE2, indicating that Ba(2+)-sensitive K+ conductance is not involved. 6. Basolateral addition of 0.1 mM 8-(4-chlorophenylthio)-cAMP inhibited only the negative VT deflection induced by PGE2. 7. The positive VT deflection was blocked by basolateral addition of 50 microM 8-(N,N-diethylamino)octyl 3,4,5-trimethoxy benzoate hydrochloride (TMB-8), an inhibitor of intracellular Ca2+ release. But elimination of luminal Ca2+ did not affect the biphasic response to PGE2. 8. These findings suggest that the initial negative VT deflection is caused by an increase in Na+ influx across the luminal membrane through an amiloride-insensitive Na+ conductive pathway, whereas the later positive deflection is caused by the inhibition of Na+ influx through the amiloride-sensitive Na+ conductive pathway. The cAMP messenger system may be responsible for the initial negative deflection, whereas an increased intercellular Ca2+ release from the store is necessary for the later positive deflection caused by PGE2. The response in the CCD is comparable to the later response in the CNT.