This study examined mechanisms of Cl transport in rat lymphocytes under a variety of conditions. Basal intracellular Cl concentration ([Cl]_i) was not different between cells assayed in the presence of HCO₃ or its absence (HEPES). Removal of external Cl resulted in a fall in [Cl]_i and a rapid rise in intracellular pH (pH_i). Both Cl efflux and the rise in pH_i were blocked by DIDS or removal of external Na⁺ but were unaffected by furosemide. The mechanisms governing Cl influx were assessed in cells that had been Cl depleted for 1 h. Reexposure to Cl resulted in a rapid rise in [Cl]_i that was partially inhibited by pretreatment with DIDS (57%) and partially inhibited by pretreatment with furosemide (45%). Pretreatment with both compounds together completely blocked Cl influx. Cl depletion caused a marked increase in pH_i that rapidly declined toward normal when the cells were reexposed to Cl. Preincubation with DIDS completely blocked this decrease in pH_i. In contrast, neither removal of Na⁺ nor preincubation with furosemide affected the decline in pH_i when the cells were reexposed to Cl. We conclude that, in thymic lymphocytes, Cl/HCO₃ (or Cl/base exchange) regulates both Cl influx and efflux. Cl efflux is totally inhibited by DIDS and is mediated by a Na⁺-dependent Cl/HCO₃ exchanger. Cl influx is partially DIDS sensitive and partially furosemide sensitive and is mediated by both a Na⁺-independent Cl/HCO₃ exchanger and by a Na⁺-K⁺-2Cl cotransporter.