Neuronal cultures derived from the septal diagonal band region of the embryonic rat brain and grown in a chemically defined medium contained a very small number of contaminating astroglial cells. During the first week in culture, these cells were well dispersed in the form of a single isolated cell with fine fibrous branched processes. Treatment with 4 microM cytosine arabinoside for 24 h failed to kill these astrocytes (most probably present in quiescent form), as judged by glutamine synthetase activity and glial fibrillary acidic protein-positive cell count. On the other hand, the exposure of cultures to cytosine arabinoside resulted in a marked increase in choline acetyltransferase enzyme activity. The overall results, together with our previous findings, are consistent with the proposal that a brief exposure to a relatively low concentration of cytosine arabinoside induces quiescent astrocytes to produce a large quantity of a neurotrophic factor that is involved in the regulation of cholinergic cells.