The ubiquitous cholinesterase (ChE) enzymes, functioning in the termination of acetylcholine mediated neural transmission, are also reported to have additional functions. Through application of siRNAs against butyrylcholinesterase (BChE) in R28 cells, a retinal cell line with pluripotent properties, a counter-regulation between ChEs was revealed. BChE knock down resulted in an up-regulation of not only acetylcholinesterase (AChE), but also altered the signaling status of PKC and ERK. Knockdown of BChE modified ERK signaling most notably through ERK1/2 proteins, together with the transcription activator P90RSK1 and c-fos. Stimulation of the R28 cell line by forskolin revealed that ChEs are involved in an intricate cross talk between different signaling pathways. Forskolin-stimulated R28 cells displayed a robust cholinergic response, as detected by both electrophysiology and ChE expression, and changed the activation status of PKC/ERK signaling pathways. The findings in R28 cells show that ChE expressions are inversely co-regulated and act through the transcription factors c-fos and P90RSK1. Since R28 cells have the capacity to differentiate into different cell types through stimulation of signaling pathways, ChEs are likely to be associated with cell fate determination, rather than just terminating cholinergic responses. (C) 2010 Elsevier Masson SAS. All rights reserved.