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In resistance arteries, matrix‐devoid holes in the internal elastic lamina (HIEL) are presumably always the site of endothelial and smooth muscle contact (myoendothelial junction; MEJs). MEJs are signaling microdomains and we have previously demonstrated that specific protein localization regulates heterocellular communication. However, it is time consuming to localize proteins at the MEJ as it requires electron microscopy (EM) verification—it also lacks quantitative analysis for spatial localization both within endothelial cells and within the tissue. Thus, we wanted to determine if every HIEL was an MEJ without relying on any single protein marker. We first developed an in‐house MATLAB analysis program to obtain quantitative data from en face images of third order mesenteric arteries, including an evaluation of spatial distribution using Monte Carlo simulations, which revealed HIEL exhibit a random spatial …