Intact, isolated nuclei and a nuclear membrane (ghost) preparation were used to study regulation of the movement of small molecules across the Xenopus laevis oocyte nuclear membrane. In contrast to models of the nuclear pore complex, which assume passive bidirectional diffusion of molecules less than 70 kilodaltons, diffusion of intermediate-sized molecules was regulated by the nuclear envelope calcium stores. After depletion of nuclear store calcium by inositol 1,4,5-trisphosphate or calcium chelators, fluorescent molecules conjugated to 10-kilodalton dextran were unable to enter the nucleus. Dye exclusion after calcium store depletion was not dependent on the nuclear matrix because it occurred in nuclear ghosts lacking nucleoplasm. Smaller molecules and ions (500-dalton Lucifer yellow and manganese) diffused freely into the core of the nuclear ghosts and intact nuclei even after calcium store depletion. Thus, depletion of the nuclear calcium store blocks diffusion of intermediate-sized molecules.