We found previously that epidermal growth factor (EGF) decreases choline acetyltransferase (ChAT) activity in forebrain cholinergic neurons in vitro indirectly via glia (Kenigsberg et al.: Neuroscience 50: 85-97, 1992). However, which glial type(s) are implicated in this response remained to be determined. Here we report that in primary cultures from the fetal rat medial septal area the complete elimination of oligodendrocytes or partial elimination of microglia from these cultures does not change the cholinergic cell response to EGF. However, the elimination of astroglia in our cultures by alpha-aminoadipic acid treatment blocks EGF's effects on the cholinergic neurons. Co-culture experiments using pure neuronal and purified glial cells from the medial septum further demonstrate that the cholinergic cell response to EGF can be maintained in the presence of astroglia only. In addition, it appears that EGF regulates the release of soluble factors from pure astroglia cultures following their peak mitotic response to EGF that decreases ChAT enzymatic activity. This soluble cholinergic neuromodulatory activity found in conditioned media from EGF-treated astrocytes has a molecular weight greater than or equal to 10 kD and loses potency following multiple freeze-thaw cycles. These results suggest that a direct glial cell response to a specific glial growth factor like EGF may have an important impact on the expression of local neurons, like the cholinergic in the forebrain.