Previous data suggested an active Cl- conductance in the renal proximal convoluted tubule, although single channel conductance and regulation were not found. We have investigated the presence and regulation of the Cl- channel in proximal convoluted tubules by patch clamp analysis. The current-voltage relationship of whole cells with 130 mM NaCl in the pipette was nonlinear. The addition of 1-34 PTH (10(-8) M), forskolin, or cAMP significantly increased whole cell Cl- conductance. We found a single Cl- channel in excised apical membranes possessing conductance of 33 picosiemens (pS) at positive and 22.5 pS at negative potential, which was blocked by 4,4'-diisothiocyanostilbene-2,2'- disulfonic acid (10(-4) M) and was selective to Cl- (Cl/Na = 10). The channel was activated by prolonged membrane depolarization, by a catalytic subunit of protein kinase A (PKA), or by purified kinase C (PKC), but not by Ca2+ (1 microM) inside the membrane. During cell-attached patch clamping, the channel was similarly activated by PTH, phorbol ester, or dibutyryl cAMP in a dose-dependent manner. To investigate second messenger contributions to the PTH-action, the PTH-evoked channels were modified further by the subsequent addition of several blockers of the second messengers. This suggested that PKA and PKC were involved in Cl- channel activation. We therefore conclude that renal proximal convoluted tubule cells possess an apical Cl- channel activated by PTH via the PKA and PKC pathways.