The intracellular pH of blood cells of the tunicate Ascidia ceratodes has been measured by equilibration of radioactively labeled markers between intra- and extracellular media. Labeled acid, 5,5-dimethyloxazolidine-2,4-dione (DMO), and base, methylamine (MA), have been used in the range of extracellular pH (pHm) of 4.5-7. For unsorted blood cells MA is less sensitive to the transmembrane pH gradient (delta pH) than is DMO in the pHm of 6.3-7. The data measured by DMO yield an intracellular pH value of 6.98 +/- 0.15. Ficoll density gradients separated 86.4% pure morula cells. Other experiments show that morula cells contain significant amounts of vanadium and most of the free tunichrome. Using both MA and DMO with morula cells yields pH values of 5.0 +/- 0.2 for the vacuoles and 7.1 +/- 0.2 for the cytoplasm. If vanadium is accumulated in the intravacuolar solution space, then this mildly acidic pH indicates that the aquo V3+ ion, which is only stable below pH 3, is stabilized by some factor other than high hydrogen ion concentration. This factor may be chelation by tunichrome. It is also possible that accumulated vanadium(III) is sequestered in hydrophobic regions of the vacuolar or cellular membranes.