5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) has been shown to produce a reduction in channel open probability in a number of epithelial chloride channels. We have investigated its action on a calcium-dependent airway epithelial chloride channel, which shows voltage-dependent gating following incorporation into planar phospholipid bilayers. Since the channel demonstrates a distinct subconductance state at approximately one-third of the fully open level, both 50 and 20% threshold analysis (TA) have been used to describe channel kinetics. Lifetime analysis using 50% TA in the absence of NPPB showed that two open and three closed lifetimes provided the optimal fit. Similar analysis using 20% TA required only two closed lifetimes and allowed for tentative assignment of tau values to either substate or fully closed events. NPPB (10-100 microM) produced a concentration-related shift of the normal voltage dependence of gating, with more negative holding potentials being required to produce a given channel open probability than in the absence of NPPB. Ten microM NPPB produced a similar shift in lifetime values. However, addition of 50 microM NPPB produced a unique, single open lifetime. No evidence was found for NPPB acting as a direct voltage-dependent blocker of chloride conductance.