1. Intracellular pH (pHi) and membrane potential (Em) of giant salivary gland cells of the leech, Haementeria ghilianii, were measured with double-barrelled, neutral-carrier, pH-sensitive microelectrodes. 2. Em was -51 +/- 11.2 mV and pHi was 6.98 +/- 0.1 (mean +/- S.D., N = 41) in Hepes-buffered saline (nominally HCO3(-)-free; extracellular pH, pHe = 7.4). pHi was independent of Em. 3. Amiloride (2 mmol l-1) had no effect on resting pHi or on pHi recovery from an acid load (induced by the NH4+ pre-pulse technique). Removal of external Na+ produced a progressive acidification which was blocked by amiloride, and the drug also slowed the recovery of pHi on reintroduction of Na+. The results indicate the presence of an electroneutral Na+/H+ exchanger whose access to amiloride is competitively blocked by Na+. 4. In certain smaller cells of the gland, which probably form a separate population, removal of external Na+ did not affect pHi, and recovery from an acid load was blocked by amiloride. There may, therefore, be two types of Na+/H+ exchanger, differing in reversibility and sensitivity to amiloride. 5. Recovery of pHi from NH4(+)-induced acid loading was not affected by bicarbonate-buffered saline (2% CO2; 11 mmol l-1 HCO3-) or by addition of the anion-exchange blocker SITS (10(-4) mol l-1). This suggests that there is no significant contribution of a HCO3(-)-dependent transport mechanism to pHi regulation in the gland cells. 6. Removal of external Cl- slowly reduced pHi and there was a transient increase (overshoot) in pHi when Cl- was reintroduced. These effects of Cl- are probably explained by changes in the Na+ gradient. Intracellular Na+ and Cl- activities were measured with ion-selective microelectrodes. 7. Acidification with NH4+ was difficult, probably because of the cells' poor permeability to this ion. Attempts to introduce NH4+ via the Na+ pump or Na+/Cl- transporter were not successful. The H+/K+ ionophore nigericin (1 microgram ml-1), however, produced a rapid and reversible acidification. 8. N-methylmaleimide (0.5-1 mmol l-1), which blocks proton-pumping ATPase, produced a prolonged acidification of almost 1 pH unit, well beyond the level expected for simple equilibration with pHe. The results are consistent with the presence of a vesicular proton pump, acidifying the secretory vesicles which pack the cell body. 9. NH4+ (50 mmol l-1) or trimethylamine (50 mmol l-1) increased pHi and stimulated salivary secretion, while propionate (50 mmol l-1) decreased pHi and stopped secretion.(ABSTRACT TRUNCATED AT 400 WORDS)