This study was undertaken to estimate the total number of cholinergic cells and the percentage of cholinergic cells that contain estrogen receptor-alpha (ER alpha) in the rat basal forebrain. Double immunostaining for choline acetyltransferase (ChAT) and ER alpha was carried out on 50-microm-thick free-floating sections. Because routine mounting method causes considerable flattening of the sections, we embedded immunostained sections in Durcupan, an epoxy resin known to cause virtually no shrinkage. When this procedure was used the section thickness was well preserved, individual cells could be clearly identified, and subcellular localization of ER alpha immunoreactivity was easy to verify. Cell counting in these sections revealed that the rat basal forebrain contains 26,390 +/- 1097 (mean +/- SEM) cholinergic neurons. This comprises 9674 +/- 504 in the medial septum-vertical diagonal band of Broca, 9403 +/- 484 in the horizontal diagonal band of Broca, and 7312 +/- 281 in the nucleus basalis. In these nuclei, 60%, 46%, and 14% of the cholinergic neurons were co-localized with ER alpha, respectively. We believe that our results are an improvement on existing data because of the better distinction of individual neurons that the Durcupan embedding method brings.