Two clonal immortalized neurons designated SN6.1b and SN6.2a were isolated by limiting dilution from a mouse embryonic septal cholinergic neuronal hybrid cell line SN6 (Hammond et al., 1986). In the serum-containing medium without extra differentiating agents, one-third of SN6.1b cells stably exhibited a morphology of differentiated neurons with extensive elaborate neurites, while a majority of SN6.2a cells, along with the parent cell line SN6, were round in shape with poorly branched short processes. Neurochemical studies showed that both clones synthesized choline acetyltransferase (ChAT), dopamine, norepinephrine, serotonin, and glutamate. Immunocytochemically, they expressed a number of neuronal antigens, such as 200-kDa neurofilament protein, neuron-specific enolase, microtubule-associated protein 2, tau protein, tubulin, neural cell adhesion molecule, Thy-1.2, saxitoxin-binding sodium channel protein, ChAT, tyrosine hydroxylase, serotonin, and glutamate. The coexistence of cholinergic, catecholaminergic, serotonergic, and glutamatergic neurotransmitter markers in the clonal hybrid septal neurons that express a variety of immunocytochemical properties of differentiated neurons suggests that embryonic septal cholinergic neurons are potentially multiphenotypic with respect to neurotransmitter synthesis.