Abstract: Rat‐1 fibroblasts were transduced to express Drosophila choline acetyltransferase. The presence of an active enzyme in these cells (Rat‐1/dChAT) was confirmed using various methods. Rat‐1/dChAT fibroblasts released acetylcholine (ACh) into the culture medium. Moreover, intra‐and extracellular levels of ACh could be increased by adding exogenous choline chloride. In addition, serum starvation or confluence‐induced quiescence caused an 80% decrease in recombinant choline acetyltransferase activity (compared with actively growing cells). ACh release was also repressed in quiescent fibroblast cultures. Exogenous choline could mitigate the decrease in ACh release. These results indicate that Rat‐1 fibroblasts can be genetically modified to produce ACh and that ACh release can be controlled by introducing choline into the culture medium. Furthermore, these data demonstrate that the expression of the retroviral promoter used in this study decreases with the onset of quiescence; however, exogenous choline can increase the amount of ACh released by quiescent fibroblasts.