Ascidians are known to accumulate high levels of vanadium. Vanadium-accumulating blood cells (vanadocytes) have one large and highly acidic vacuole. Recently, it was found unexpectedly that the number of vanadocytes increased rapidly and significantly when ascidians were immersed in 10 mM or 20 mM NH₄Cl solution for 20 hr to neutralize vacuole content. Suspecting that changes in intra-organellar pH and in levels of ATP caused by the treatment might be involved, we examined whether or not several reagents that perturb either acidic pH or ATP synthesis affected the increase in the number of vanadocytes. SF6847 (a proton conductor), nigericin, monensin, valinomycin (ionophores), 2,4-dinitrophenol (an uncoupler), bafilomycin A₁ (a V-ATPase inhibitor), oligomycin and NaN₃ (F-ATPase inhibitors) all increased the number of vanadocytes by about three- to five-fold over that of control. However, treatment with NaCl, KCl, LiCl, CaCl₂, TJ24373, sporeamycin (macrolide antibiotics), ouabain and Na₃VO₄ (P-ATPase inhibitors) had no effect on the increase. These results suggest that neutralization of intra-organellar pH triggers an increase in the number of vanadocytes. Vanadocytes that increased in number in the coelomic fluid after treatment were revealed by immunohistochemical study, to have originated in the connective tissues around the alimentary canal.