The compound 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) is being extensively used to measure intracellular pH (pHi) in a variety of tissue preparations. Use of this dye in the isolated perfused rabbit cortical collecting tubule (CCT) revealed two previously unreported findings. Apical incubation via luminal perfusion with the acetoxymethyl ester of BCECF (BCECF-AM) resulted in intense uptake into a minority cell population. Basolateral incubation via the peritubular bathing solution resulted in apparent homogenous uptake into all cells. The minority cells were identified as intercalated cells by the use of fluorescent probes specific for intercalated cells. Minority cell pHi was 7.34 +/- 0.06 (n = 9); majority cell pHi was 7.41 +/- 0.05 (n = 6). In addition, prolonged excitation of BCECF resulted in adverse effects. Intense continuous excitation for 1-3 min resulted in a fall in apparent pHi of 0.53 +/- 0.05 pH units (n = 6, P less than 0.001). After reloading with fresh BCECF-AM, the calculated pHi was 0.05 +/- 0.07 pH units higher than before prolonged excitation (P = NS), suggesting that the apparent decrease was due to changes in the pH sensitivity of BCECF as a result of the prolonged excitation. Prolonged excitation of cells with pHi clamped at 7.50 resulted in a decline in apparent pHi at the rate of 0.15 +/- 0.02 pH units/min (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)