A method for treating isolated hepatocytes with a known and toxicologically meaningful concentration of CCl4 was used to study the effect of the haloalkane on the calcium sequestration capacity of microsomal vesicles derived from the hepatocytes. The essence of the method is to allow a very small volume of CCl4 to come to diffusion equilibrium in a closed system characterized by a gas phase that is large relative to the aqueous phase in which hepatocytes are to be suspended. The final equilibrium concentration of CCl4 in the aqueous phase is 174 microM. When hepatocytes are added to the aqueous phase, after at least 1 hr of incubation of the resealed system at 37 degrees C, the cells do not liberate GOT to the medium above control values, and their capacity to exclude trypan blue is unimpaired. However, covalent binding of the 14C from 14CCl4 is maximal within the first 10 min of incubation, and the calcium sequestering capacity of microsomal vesicles derived from hepatocytes incubated in the presence of CCl4 is reduced to 30% of control levels after 20 min. This system should prove useful in study of early effects of CCl4-dependent derangement of hepatocellular calcium homeostasis.