1. The intracellular Cl- concentration, [Cl-]1, of rat renal outer medullary slices has been studied in iso-osmolal media containing 42, 93, 144, 189 or 225 mM chloride, [Cl-]0. Equilibrium values for [Cl-]1 were attained within 25-50 min from the start of incubation such that [Cl-]1/[Cl-]0 = 0-46. This ratio was independent of [Cl-]0 within the range studied. Intracellular Na+ was unaffected, and only minor variations of cell volume were observed (calculated from slice weight changes and [14C]carboxyl inulin spaces. 2. When [Cl-]0 = 189 mM, [Cl-]1 remained constant at 87 mM for up to 50 min, indicating that these figures may represnet the interstitial [Cl-] and mean intracellular [Cl-] respectively in outer medulla. 3. Omission of bicarbonate from medium containing 189 mM-Cl- caused an increase in [Cl-]1/[Cl-]0 to 0-58, which was not significantly affected by anoxia or by the presence of arsenite (5 X 10(-3) M) or 2,4-dinitrophenol (10(-3) M). Significant further increases were observed in the presence of iodoacetic acid (5 X 10(-3) M) (0-70), acetazolamide (10(-3) M or 5 M 10(-3) M) (0-71) and iodacetic acid plus 2,4-dinitrophenol (0-85). The addition of the diuretic agents ouabain, ethacrynic acid-cysteine and frusemide (all 10(-3) M) to 189 mM-Cl- media containing bicarbonate reduced [Cl-]1/[Cl-]0 to 0-36, 0-37 and 0-42 respectively. 4. The mean 36Cl- space of outer medulla after 50 min incubation in 189 mM Cl- medium was 49-7 +/- 2-1 micronl./100 mg wet wt. The volume of distribution was not significantly affected by ouabain, ethacrynic acid-cysteine or frusemide (10(-3) mM). 5. Net efflux of 36Cl- from slices loaded with isotope into 'cold' 189 mM Cl medium showed three components with rate constants of 69 X 10(-3), 18 X 10(-3) and 9-9 X 10(-5) sec-1 respectively. Efflux was not affected by ouabain, ethacrynic acid-cysteine or frusemide (10(-3) mM). 6. The main conclusions drawn from this study are: (i) the interstitial fluid Cl- concentration of normally hydrated rat outer medulla is approx. 189 mM; (ii) the [Cl-]1/[Cl-]0 ratio 0-46 may represent a chiefly passive distribution maintained by the opposing gradient of a second anion, probably bicarbonate; (iii) the energy required to maintain unequal distribution of Cl- in the absence of external bicarbonate is derived chiefly from glycolysis, with a small aerobic component; (iv) there may be two intracellular chloride pools whose net rates of Cl- exchange differ by a factor of approx. 180.