Regulation of the intracellular pH (pHi) of normal rat lactotrophs was studied. As this cell type, cultured with 10% FCS, can achieve a relatively alkaline pHi (7.3-7.5), we investigated the presence of a mechanism based on Cl-/HCO3- exchange. Using the pHi-sensitive probe SNARF-1 (seminaphtorodafluor) in its permeant form, SNARF-1/AM, we studied pHi recovery after acidic loading in individual cells with a microspectrofluorometric approach. We showed the involvement of anionic exchange in lactotroph cell pHi regulation. Acute CO2-bicarbonate cell acidic loading combined with external Cl- depletion induces the activation of a Cl-/HCO3- exchange. This exchange is 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitive and corresponds to the type 3 anionic exchanger (AE3). However, after nigericin acidification, Na+/H+ exchange can also participate in recovery. In addition, incubation experiments strongly suggest that a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive anionic exchanger (type 2 anionic exchanger or AE2) is present in rat lactotrophs. The presence and involvement of carbonic anhydrase in pHi regulation have been demonstrated. Finally, using Northern blot and reverse transcription-PCR techniques, messenger RNAs for both AE2 and AE3 were identified in anterior pituitary cell extracts. We concluded that in normal rat lactotrophs, pHi regulation is achieved by a complex system in which Cl-/HCO3- exchange has a pivotal role.