Immunocytochemistry of choline-acetyltransferase (ChAT) has been combined with autoradiography of [
3H]choline after retrograde axonal transport on the same tissue section. This combination was performed with efficient preservation, in situ, of [
3H]choline or its derivatives by ionic fixation with silicotungstic acid. The cholinergic neuronal pathway from the diagonal band of Broca (DBB) of basal forebrain to the olfactory bulb (OB) was investigated with this method. After the injection of [
3H]choline into the OB, radiolabelled neurons were identified in the DBB. Most of them were ChAT immunoreactive confirming the specificity of [
3H]choline retrograde labelling for cholinegic neurons. At the electron microscopic level, silver grains could be observed only on the cytoplasm. This method will be useful to study the coexistence of transmitters in the cholinergic pathways and at the same time to determine the chemical nature of the afferences making contact with a defined soma of a cholinergic pathway.
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