The intracellular pH (pHi) dependencies of the Na-H exchanger and also of unidentified acid-loading processes ("H delivery") in IEC-6, a rat intestinal crypt cell line, were determined using microspectrofluorimetry of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein on groups of cells grown on glass cover slips mounted in a perfusion chamber in HCO3-free solutions (to inactivate Cl-HCO3 exchange and Na-HCO3 cotransport). Na-H exchange and H delivery rates were measured using two methods. 1) Cells were acid loaded with NH4Cl, and rates of pHi recovery and acidification were measured just before and then just after adding amiloride at various times during the recovery. Multiplying rates of pHi recovery times the intrinsic buffer capacity (beta i) yielded net H efflux rates, rates of pHi decrease (during amiloride treatment) times beta i yielded H delivery rates, and Na-H exchange equals net H efflux plus H delivery. 2) In control cells, time courses of amiloride-induced pHi decreases (from 7.36 to 6.65) and of pHi recovery following amiloride removal (from 6.65 to 7.49) were curve fit by computer; rates of pHi decrease and recovery were calculated; and H delivery, net H efflux, and Na-H exchange were then calculated at the different pHi as above. H delivery and Na-H exchange were both pHi dependent, but in the opposite directions. H delivery was 2-7 mM/min at the control pHi, decreasing to zero around pHi 6.6. Na-H exchange was also equal to 2-7 mM/min at the control pHi, and this increased as pHi decreased; at pHi 6.6, Na-H exchange was 20-35 mM/min.(ABSTRACT TRUNCATED AT 250 WORDS)