Replacement of perfusate Cl- with Br- reduced by 37% the volume of saliva secreted by isolated, perfused rat submandibular glands exposed to 10(-6) M acetylcholine. Replacement of perfusate Cl- with SO4(2-) reduced fluid secretion by 73%, while replacement of bicarbonate with HEPES or addition of acetazolamide had no effect on salivary flows or volumes. Furosemide (10(-3) M) reduced saliva secretion when added to Cl- -(73%) or Br- -containing (56%) perfusates and also to HEPES- (95%) or acetazolamide-containing (82%) perfusates. Perfusion of the glands with furosemide or SO4- containing perfusates resulted in significantly reduced salivary Cl- concentrations and increased residual anion (Na+-K+-Ca2+-Cl-) concentrations, while perfusion with solutions containing acetazolamide or with HCO3- -free (HEPES) perfusates resulted in opposite changes in salivary anions. Addition of furosemide to HEPES- or acetazolamide-containing perfusates reversed these changes and caused a reduction in salivary Cl- concentrations and an increase in residual anion concentrations. It is concluded that 1) extracellular Cl- is required for saliva secretion in the rat submandibular gland, 2) the required external Cl- enters the salivary cells by a furosemide-sensitive transport system, which is likely to be similar to a Na+-K+-Cl- cotransport system described in other epithelia, 3) HCO3- is not normally required for saliva secretion but can sustain it partially when Cl- transport is inhibited by either furosemide or Cl- replacement, and 4) salivary electrolyte changes induced by Cl- or HCO3- replacement or by furosemide probably represent combined effects on acinar secretion and transductal electrolyte transport.