Chronic excitotoxicity mediated through defective glial and/or neuronal glutamate transport may contribute to several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). This study was performed to determine the ultrastructural characteristics of astroglial changes concomitant with motor neuron (MN) degeneration in a model of slow excitotoxicity in vitro. The study was performed on organotypic cultures of rat lumbar spinal cord subjected to the glutamate uptake blockers threohydroxyaspartate (THA) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC). The chronic inhibition of glutamate transport by THA and PDC resulted in slow degeneration of the rat's MNs accompanied by distinct glial changes predominantly involving protoplasmic astrocytes. The presence of irregular vacuoles and vesicles in the astroglial cells was frequently observed. Occasionally the astrocytes exhibited proliferation and accumulation of abnormal profiles of smooth endoplasmic reticulum. In 3 weeks there were no signs of increased production of glial filaments in the protoplasmic astrocytes. The results evidenced the coexistence of neuronal degeneration and astroglial abnormalities in an ALS model in vitro and suggested an active role of astrocytes contributing to the induction and propagation of MN degeneration.