The temporal and spatial changes of intracellular free calcium ([Ca2+]i) within the cytosol and nucleis of C6 glioma cells have been investigated with laser confocal scanning microscopy to evaluate the current view that Ca2+ ions pass freely through nuclear pores by diffusion. Our results indicate that localized cytosolic Ca2+ release, which appeared as puffs, spread with an apparent diffusion rate of 0.35 +/- 0.07 microns/sec (n = 44). This release was followed by an immediate Ca2+ uptake at the resting stage. Following the treatment with thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, release of nuclear Ca2+ from certain nuclear hot zones and nuclear envelope was obtained. Most of the nuclear Ca2+ released were confined to the nuclear boundary, but a slow migration of Ca2+ towards the cytosol was observed. The apparent diffusion rate of this Ca2+ release is 0.015 microns/sec. By contrast, the inward spread into nucleus occurred with a diffusion rate of 0.04 microns/sec. From these diffusion rates and other experimental evidence, we conclude that the movement of Ca2+ at the nucleocytosolic interface is more than a simple diffusion process and the interface is a barrier to Ca2+ movement.