Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) have previously been shown to increase the survival and/or differentiation of cholinergic spinal motoneurons in culture. We report here that CNTF and LIF increase choline acetyltransferase (ChAT) activity three- to fourfold in cultures from the ventral mesencephalon (VM) containing cholinergic neurons from cranial motor nuclei and the pedunculopontine nucleus, but decrease tyrosine hydroxylase activity to 50-60% of control values. In contrast, they do not increase cholinergic properties in cultured septal and striatal cholinergic neurons. In order to identify the subpopulations of cholinergic neurons in the mesencephalic cultures responding to CNTF and LIF, embryonic VM was cut into rostral, caudal, central, and laterocaudal portions and cultured separately. CNTF and LIF increased ChAT activity only in the rostral and central VM, which contain cranial motor nuclei, and not in the caudal VM, which contains the rostroventral part of the pedunculopontine nucleus. Together with the previous observations on spinal motoneurons, the present results indicate that only cholinergic motoneurons projecting to peripheral targets, but not cholinergic neurons projecting centrally, may respond to CNTF and LIF by an increase in ChAT activity or by increased neuronal survival.