The development of Drosophila midline glia during larval and pupal stages was characterized by localizing beta-gal expression in enhancer trap lines, as well as with BrdU incorporation and pulse-chase experiments. At hatching about 40 to 50 glial cells are present along the midline of the ventral nerve cord (2 to 3 dorsal and 1 to 2 ventral cells per neuromere). The cells proliferate during the third larval instar and spread dorsoventrally within the midline, increasing in number to about 230 or more (around 20 cells per neuromere). Cell divisions cease shortly after pupariation, and the cells persist for the first half of pupal life with no apparent changes in numbers or positions. Between 50 and 80% of metamorphosis, however, virtually all of the midline glia undergo programmed cell death. Tissue culture experiments indicate that the peak of ecdysteroids occurring at pupariation is required for the cessation of proliferation of midline glia and their subsequent degeneration. Midline glia in central nervous systems (CNS) cultured with low or no ecdysteroids survive and continue to divide, whereas they cease proliferating and later degenerate with high ecdysteroids levels. The midline glial may play a role during CNS metamorphosis similar to that of their progenitors in the embryo, in stabilizing outgrowing neurites that cross or run along the midline.