The collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase (AChE) in vertebrate muscles. Two ColQ transcripts, ColQ-1 and ColQ-1a, driven by two distinct promoters are expressed differentially in mammalian slow- and fast-twitch muscles, respectively. Such expression patterns are determined by the contractile activity in different muscle fiber types. To reveal the regulatory role of muscular activity on ColQ expression, acetylcholine and nicotine were applied onto C2C12 muscle cells: the challenge increased the expression of ColQ-1/ColQ-1a mRNAs. The agonist challenge induced the phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In parallel, over expression of an active mutant of CaMKII enhanced both ColQ-1/ColQ-1a mRNA levels in cultured C2C12 myotubes. Moreover, the over expression of myocyte enhancer factor 2 (MEF2), a downstream mediator of CaMKII, in the myotubes potentiated the CaMKII-induced ColQ expression. The current results reveal a signaling cascade that drives the expression profiles of ColQ in responding to activity challenge in cultured myotubes.