The rate of acetylcholine (ACh) synthesis was found to depend on the activity of choline acetyltransferase (ChAT) and on the concentrations of the two substrates of this enzyme, choline and acetyl-CoA. In SN56 cells treated for 3 days with 1 mM dbcAMP activities of ChAT and acetylcholinesterase (AChE) were elevated. It was accompanied by an increased activity of ATP-citrate lyase (ACL)-an enzyme responsible for provision of part of acetyl-CoA for ACh synthesis in cholinergic neurons. In contrast lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) activities were reduced by dbcAMP. Treatment with 0.001 mM all-trans retinoic acid (RA) elevated ChAT and LDH activities but reduced the activities of AChE and ACL. The combined treatment with db-cAMP and tRA increased ChAT activity in supraadditive fashion. The effects of these two compounds on the other enzymes were not additive. Neither compound altered the activities of carnitine acetyltransferase, acetyl-CoA synthase, or acetyl-CoA hydrolase. On the other hand, they decreased acetyl-CoA content and rate of ACh release. Overall, the results indicate that tRA upregulates only ChAT expression, whereas dbcAMP upregulates several features of cholinergic neurons including ChAT, AChE, and ACL. Low levels of acetyl-CoA in differentiated cells may result in a low rate of ACh release and resynthesis during their depolarization.