Different classes of glia cells in the optic lobes of Drosophila melanogaster were defined by the enhancer trap technique, using expression of the lacZ reporter gene. At both the outer and inner optic chiasms, there are stacks of glia, arrayed from dorsal to ventral, interpersed between the crossings of axonal fiber bundles. The giant glial cells of both the outer and inner chiasms are similar with respect to their nuclear shapes and positions, indicating similar functions of these cell types. Another class of glia is found in the medulla neuropil. Their cell bodies anchor in the most distal region of the neuropil, and their processes extend into the deeper neuropil layers. Birth dating using BrdU shows that both groups of chiasm glia are born early in larval life; they may participate in the development of the optic lobe. The medulla glia are born later and may be involved primarily in adult functions. In the wild type, and in mutants with structurally altered optic lobes, the numbers of tract-associated glial cells in the outer and inner optic chiasms seem to vary with the number of visual columns, whereas the complement of medulla neuropil glia correlates with the volume of the optic lobe.