Stem cells are used to generate differentiated somatic cells including neuronal cells. Synthesis and release of acetylcholine, a neurotransmitter and widely expressed signalling molecule, were investigated in the murine embryonic stem cell line CGR8 during early differentiation, i.e. in the presence of leukaemia inhibitory factor (LIF) to maintain pluripotency and in the absence of LIF to induce early differentiation. CGR8 cells express choline acetyltransferase (ChAT) as demonstrated by measurement of enzyme activity and substantial inhibition by bromoacetylcholine. Pluripotent CGR8 cells showed a ChAT activity of 250pmol acetylcholine/mg/h, contained 1.1pmol acetylcholine/10(6) cells and released about 12.00pmol acetylcholine/1×10(6) cells/6h. Removal of LIF induced early differentiation as evidenced by reduced transcription factors Oct-4 and Nanog and a substantial slowing of the proliferation rate. Under this condition acetylcholine synthesis increased to 1640pmol/mg/h; related to the pluripotent state the content of acetylcholine increased 10fold and the release to about32pmol acetylcholine/1×10(6) cells/6h.Enzyme kinetic analysis showed a significant increase of the Km for the precursor acetyl-CoA and of Vmax without a change of the Km for the precursor choline. In conclusion, early differentiation of the stem cell line CGR8 is associated with a substantial increase in ChAT activity and acetylcholine release.