We measured fluoride flux (JF; pmol ·; min-1 · mm-1) in the isolated rabbit cortical collecting duct (CCD) to investigate the determining factors of JF. The perfusate contained 100 μM fluoride and the bath was fluoride-free. Osmotically-induced lumen-to-bath water flux did not affect JF. When perfusate pH was reduced from 7.4 to 6.1 and from 6.1 to 5.0, JF increased from 0.008 ± 0.002 to 0.027 ± 0.007 (P < 0.01) and from 0.018 ± 0.003 to 0.040 ± 0.005 (P < 0.01), respectively. Acetazolamide at 10-4 M in the bath reduced JF slightly though not statistically. The anion-transport inhibitor, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS), at 10-4 M in the perfusate did not affect JF. Substitution of luminal chloride with gluconate failed to affect JF in tubules from normal rabbits or from rabbits treated with deoxycorticosterone which stimulates chloride-bicarbonate exchange in the CCD. JF showed no correlation with transepithelial voltage which ranged from +4 to -104 mV. We conclude that the luminal pH represents the primary determining factor influencing JF in the rabbit CCD, and fluoride does not use a chloride-mediated or a DIDS-inhibitory transport pathway.