The onset of spontaneous contraction in rat primary muscle cultures coincides with an increase in acetylcholinesterase (AChE) activity. In order to establish whether contractile activity modulates the rate of AChE transcript synthesis, and what elements of the gene are determinant, we examined the promoter and intron I in contracting muscle cultures. Ache genomic fragments attached to a luciferase reporter were transfected into muscle cultures that were either electrically stimulated or paralyzed with tetrodotoxin to enhance or inhibit contractions, respectively. Cultures transfected with intron I-containing constructs showed a 2-fold increase in luciferase activity following electrical stimulation, compared to tetrodotoxin treatment, suggesting that this region contains elements responding to contractile activity. Deleting a 780 bp distal region within intron I, containing an N-box element at +890 bp, or introducing a 2-bp mutation within its core sequence, eliminated the contraction-induced response. In contrast, mutating an N-box element at +822 bp had no effect on the response. Furthermore, co-transfecting a dominant negative GA-binding protein (GABP), a transcription factor known to selectively bind N-box elements, reduced the stimulation-mediated increase. Our results suggest that the N-box within intron I at +890 bp is a regulatory element important in the transcriptional response of Ache to contractile activity in muscle.