A two-color fluorescence method is described for demonstrating immunohistochemically the anterogradely transported plant lectin Phaseolus vulgaris leucoagglutinin (PHAL, fluorescein isothiocyanate label) and choline acetyltransferase (ChAT, rhodamine label) on the same rat brain section. Application of this method to the study of projection neurons in the vertical and horizontal limbs of the diagonal band, the substantia innominata and nucleus basalis revealed that both cholinergic and non-cholinergic pathways followed similar trajectories to their targets. These included: projections from the vertical, and, to a lesser extent, horizontal limb of the diagonal band coursing through the dorsal fornix, alveus and fimbria to the hippocampus; fibers from the vertical and horizontal limbs of the diagonal band traveling anteriorly to the anterior olfactory nucleus, posterolaterally to the entorhinal cortex, and anterodorsally into the cingulum to the cingulate and retrosplenial, and, in some cases, the frontal and occipital cortices; projections, mostly non-cholinergic, from the substantia innominata traveling laterally to the piriform cortex and amygdala, and anteriorly to the anterior olfactory nucleus and olfactory bulb; and fibers from cells in the nucleus basalis coursing dorsally to the frontal and parietal cortices or laterally to the basolateral amygdala and piriform, insular and temporal cortices. Some axon terminations ended at right angles to the parent axon shaft in short protuberances resembling terminal boutons.