Glial fibrillary acidic protein (GFAP)-positive astrocytoma cells were stably transfected with an expression vector carrying a murine complementary DNA for GFAP in the antisense orientation. Three stably transfected GFAP-negative transformants were identified by indirect immunofluorescence and expanded in vitro. The stably transfected and control cell clones were analyzed for morphological alterations, growth in monolayer and soft agar, adhesiveness, and in vitro invasive potential. In contrast to control astrocytoma cells which retained an astrocytic phenotype with polygonal or triangular cells extending multiple long and thin processes, the antisense GFAP-transfected cells demonstrated marked morphological alterations in the form of flat, epithelioid cells devoid of long, astrocytic glial processes. The antisense GFAP-transfected clones demonstrated a greater degree of cell crowding and piling at confluence than did controls. By tritiated thymidine analysis, the antisense GFAP-transfected cell clones demonstrated a 2-3-fold increase in incorporation of the radiolabel, suggesting an enhanced proliferative potential over controls. Antisense GFAP-transfected astrocytoma clones formed larger and more numerous colonies than did controls when tested for anchorage-independent growth in soft agar. Following a time-course adhesion assay, antisense GFAP-transfected astrocytoma clones were found to be less adherent to their substratum than controls. When assessed in an in vitro invasion assay system, antisense GFAP-transfected astrocytoma cells more readily penetrated Matrigel-coated filters than did controls. These data have shown that eliminating GFAP expression from astrocytoma cells has affected astrocytoma cell morphology and adhesion. The data also suggest that the growth and invasive potential of the antisense GFAP-transfected astrocytoma cells have been significantly enhanced by altering the expression of this glial-specific cytoskeletal protein in this experimental cell system.