Using polyclonal and monoclonal antibodies to visualize under a confocal microscope type-1 cannabinoid receptors (CB1) and acetylcholine (ACh) receptors, respectively, or α-bungarotoxin conjugated to Alexa-Fluor 555 for Ach receptors, we found that they colocalize on twitch muscle fibers in the frog (Rana pipiens). We show that both the CB1 and ACh receptors are present on the fast skeletal muscle motor end-plate. The CB1 receptor is present along the entire membrane of the muscle fiber, whereas the ACh receptor is expressed primarily at the motor end-plate. Analysis of the colocalization produced a cross-correlation coefficient of 0.519 ± 0.021 (n = 9) for both receptors at the muscle motor end-plate. This study suggests a close proximity between these two types of receptor proteins and that they could interact. CB1 could function at some stage of excitation-contraction coupling in these muscle fibers. However, further investigation is needed in order to clarify these issues.