Mice carrying copies of the human glial fibrillary acidic protein (hGFAP) gene driven by its own promoter have been generated that express the human transgene at different levels (Messing et al.: 152: 391–398, 1998). Lines that expressed high levels of the gene died shortly after birth. Astrocyte cultures prepared from a low overexpressor (Tg73. 2) exhibited abnormal cytoplasmic inclusions identical to those seen in vivo in the high overexpressors. Astrocytes in the Tg73. 2 cultures appear odd-shaped and enlarged, express increased levels of GFAP (both human and mouse), and express αB crystallin protein, Hsp27 protein, and vimentin protein. At the light microscopic level, the Tg73. 2 astrocytes are filled with eosinophilic deposits surrounded by positive GFAP immunostain. Ultrastructurally, the Tg73. 2 astrocytes contain osmophilic deposits on a bed of intermediate filaments identical to Rosenthal fibers found in the brain in Alexander's disease. It seems that Tg73. 2 mouse astrocytes in culture do not require additional stress from external sources or contact with other neuroectodermal cells to produce Rosenthal fibers. This suggests that the added hGFAP gene is sufficient to induce Rosenthal fibers and that an excess of GFAP in astrocytes may be detrimental to normal function. We hypothesize that the normal mechanism for GFAP turnover may be insufficient to handle the excess GFAP, thus causing an accumulation of stress proteins. The increased amounts of stress proteins and GFAP results in the formation of Rosenthal fibers, similar to those found in Alexander's disease. J. Neurosci. Res. 53: 353–360, 1998.© 1998 Wiley-Liss, Inc.