The 2068 clone of the PC12 rat pheochromocytoma cell line, which has higher intracellular D-Asp levels than the PC12 cell line, was investigated for the presence of D-aspartate (D-Asp) by employing the anti-D-Asp antibody that we had previously developed. Confocal laser scanning fluorescence microscopy revealed that D-Asp immunoreactivity (IR) is present in granule-like structure(s), which suggests that D-Asp is associated with vesicles within the cells. Depolarization by high KCl concentrations and a Ca²⁺ ionophore increased D-Asp release from the cells. This release was significantly suppressed by depleting extracellular Ca²⁺ ions, which suggests that D-Asp is released from the cells through exocytotic processes. D-Asp IR in 2068 cells is also prominent in round-shaped cells that appear to be undergoing mitosis. The immunofluorescence intensity of D-Asp at every confocal plane was integrated throughout the entire depth of each cell and compared between the round-shaped mitotic cells and the flat resting cells. Total fluorescence intensities in the mitotic cells are higher than those in flat resting cells, suggesting that cellular D-Asp levels may be related to the cell cycle of 2068 cells.