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1. The steady-state intracellular/extracellular concentration ratios (Ci/Co) of a number of radiolabelled weak bases in isolated rat superior cervical ganglia were measured. 2. Observed values for Ci/Co (mean +/- S.E. of mean) were [3H]nicotine, 6.17 +/- 0.12; [14C]morphine, 6.08 +/- 0.14 [3H]atropine, 7.10 +/- 0.16; [14C]trimethylamine, 6.73 +/- 0.13; [14C]procaine, 10.13 +/- 0.26. If Ci/Co were determined by the transmembrane pH gradient, the intracellular pH (pHi) appropriate to these concentration gradients lay between 6.4 and 6.6 at an extracellular pH (pHo) of 7.4. 3. the steady-state value of Ci/Co for the weak acid 5,5-dimethyl-2,4-oxazolidinedione (DMO) was 0.87 +/- 0.007. The appropriate pHi was 7.31 +/- 0.003. 4. The difference between the values of pHi calculated from the distribution of the weak bases and of DMO could not be attributed to (i) experimental error, (ii) partial permeation of protonated base, (iii) intracellular binding or carrier-mediated transport of base, (iv) lipid uptake of base or (v) different pK'a inside and outside cells. 5. The difference between the measurements of pHi made with DMO and nicotine (pHDMO-pHnic) was reduced or abolished by uncoupling agents, which act as transmembrane proton carriers. This effect was not reproduced by respiratory inhibitors or by exposure to lactate. 6. pHDMO-pHnic was small (less than 0.1 units) in human erythrocytes, which contain no intracellular organelles, and was exaggerated (1.0 unit) in slices of lipid-depleted brown adipose tissue which contained an abundance of mitochondria. 7. It is concluded that the different values of pHi determined using weak acids and bases arise from the presence of membrane-bound intracellular compartments of differing pH, and that where the use of pH-sensitive micro-electrodes is impracticable, it is desirable to measure pHi with both a weak acid and a weak base unless these can be shown equal over a wide range of pHi values.