Many neurons in the medial septal nucleus lose their transmitter-associated enzyme staining following axotomy in the proximal fimbria-fornix (FF), but it is not clear if these neurons have died or persist in a shrunken and subfunctional state. To investigate this further, septal neurons projecting through the FF were labelled with the fluorescent dye, True blue, by retrograde transport from multiple bilateral injection sites in the hippocampus. True blue-labelled neurons and cholinergic neurons immunohistochemically stained for choline acetyltransferase (ChAT) were then quantitatively compared in neighbouring sections through the medial septum 28 days after complete unilateral transections of the proximal FF. The number of True blue and ChAT positive cells ipsilateral to the FF lesion showed significant (P less than 0.001) declines of 51.4% and 71.1%, respectively, relative to the unlesioned side. Cell loss was considerably more severe among large neurons, such that 78.0% and 92.7% of True blue and ChAT labelled cells larger than the normal mean, but only 40.1% and 68.0% of True blue and ChAT labelled cells smaller than the normal mean size were lost. This indicates either that larger neurons were more prone to cell loss, or that some (but not all) large neurons persisted in a shrunken form. Histograms showed no increase in cell number in any of the smaller size categories and a substantial decrease in most cases, indicating that shrinkage alone could not account for the loss of all large neurons. Since True blue can remain present in brainstem cholinergic neurons surviving for over 365 days after axotomy, loss of True blue suggests breakdown of membrane integrity and cell death.(ABSTRACT TRUNCATED AT 250 WORDS)