pChAT is a splice variant of a peripheral type encoded alternatively by the gene for choline acetyltransferase of the common type (cChAT), the enzyme responsible for acetylcholine synthesis. Immunohistochemistry using pChAT antiserum has successfully visualized many known peripheral cholinergic cells, whereas most cChAT antibodies failed to do so. As, however, accumulating evidence indicates that pChAT expression also occurs in various non‐cholinergic neurons, we examined possible acetylcholine production by pChAT in rat dorsal root ganglion as a model. The present study indicated that the ganglion neurons possessed pChAT, but never cChAT, mRNA and protein. Our detailed analysis further showed that, despite low enzyme activities of both choline acetyltransferase and acetylcholinesterase, the level of acetylcholine in the ganglion was as high as to that in various brain regions receiving cholinergic innervation. By using immunoprecipitation methods, we here provide evidence that pChAT definitely has enzyme activity enough to supply physiological concentrations of acetylcholine in the ganglion. We propose that pChAT contributes both to acetylcholine neurotransmission in physiologically identified cholinergic cells and to functions yet unknown in non‐cholinergic neurons. Thus pChAT provides a new window on the role of neuronal acetylcholine.