Previous studies have shown that disulfonic stilbenes, arylaminobenzoates, sulfonylureas, salicylates, and sulfonamide diuretics block CFTR. These structurally diverse compounds all possess at least one negatively charged group and aromatic ring. Phenol red, which is used widely as a pH indicator in cell culture, also has these features. Thus the aim of the present study was to determine if phenol red can block CFTR. Excised inside-out patches were obtained from L cells that stably express human wt-CFTR. PKA and ATP were added to maintain CFTR channel activity. Addition of phenol red to the bath solution caused CFTR to undergo frequent transitions to a non-conducting (blocked) state in records containing a single level of channel activity and reduced the mean current amplitude of multi-channel recordings. Channel activity was restored following a serial dilution of the bath with an identical solution containing no phenol red. Analysis of the reduction in mean current revealed that 170±50 μM phenol red will cause a 50% inhibition (n = 3). Since cell culture media contains 5-21 mg/l of phenol red (13-56 μM) the open probability of CFTR may be reduced up to 25% in cell-attached patches. Thus culture media formulated without phenol red should be considered in future studies of CFTR channel activity.