Recombinant green fluorescent protein (GFP)-tagged Eps15 homology domain-containing (EHD) protein localizes to the neuromuscular junction (NMJ). Control and EHD1 proteins were expressed for 3 or 14 days in 6- to 10-week-old C57Bl/6 J mouse tibialis anterior (TA) skeletal muscle as C-terminal and N-terminal GFP recombinant proteins. Four mice were injected for each recombinant protein expressed. The TA muscle was fixed in situ and teased into small bundles for immunofluorescence and visualization via confocal microscopy, referenced to α-bungarotoxin (BTX) labeling of AChRs at the NMJ. Expression of GFP (vector) was seen as a diffuse signal throughout the myofiber and did not concentrate at the NMJ (n = 7). Rapsyn-GFP expression localized exclusively to the NMJ of transfected fibers (n = 2). Both transfected and non-transfected fibers are shown in the bottom left panel. N- and C-terminal GFP recombinant EHD1 proteins localized to the NMJ and to the primary synaptic cleft with BTX after 3 or 14 days of transfection (n = 24). Each image consists of separate blue (DAPI), green (BTX Alexa Fluor 488 conjugate), and red (anti-GFP Alexa Fluor 555 conjugate) channels in addition to a merged image. Scale bars: 20 μm. The larger reference bar was added in powerpoint to make the 20 μm bar visible in the images. It reflects the 20 μm scale bar that was added within the Zen software upon image acquisition.