The ability of embryonic day 12 and 13 optic tectum cells to replace depleted A2B5(+) cells and neurons was tested by immunomagnetic cell separation. Nearly all purified surface A2B5(-) cells were identified as glia by immunoreactivity for either glutamine synthetase of galactocerebroside. Most (approximately 80%) of the purified A2B5(-) cells became A2B5(+) after 1 day in culture, although no increase in the percentage of A2B5(+) cells (from 45%) was observed in control cultures of unpurified cells. Long-term monolayer cultures from purified cells contained A2B5(+) cells with mostly flattened glial-like or round process-free morphology, whereas those from unpurified cells contained many A2B5(+) neurons. The non-neuronal A2B5(+) cells frequently reacted with antibodies against glial fibrillary acidic protein and another marker expressed by embryonic brain glia, 5A11. Additionally, some flattened glia-like cells exhibited elaborate networks of anti-neurofilament-M-reactive filaments. We believe these unusual phenotypes, which appeared only in cultures of purified A2B5(-) cells, arose in response to the immunomagnetic removal of neurons. In conjunction with previous findings, we conclude that the abnormal phenotypes in purified cell cultures represent glia that were unsuccessful in attempting to replenish the depleted neuronal population. This may reflect restricted developmental potentials that arise during brain ontogeny.