OBJECTIVE: The primary aim of this study was to evaluate the effectiveness of laser speckle contrast imaging (LSCI) coupled with transdermal iontophoretic delivery of acetylcholine (ACh) for the assessment of penile microvascular function. Additionally, we tested systemic microvascular function using both LSCI and ACh iontophoresis on the forearm. METHODS: We assessed cutaneous, endothelium-dependent microvascular reactivity on the penis and forearm of healthy volunteers (aged 56.6 ± 1.0 years, n = 26) at rest and 60 min following the oral administration of the phosphodiesterase type 5 inhibitor (sildenafil: SIL, 100mg). LSCI was coupled with the iontophoresis of ACh using increasing anodal currents of 30, 60, 90, 120, 150 and 180 μA during 10-second intervals spaced 1 min apart. RESULTS: Basal skin microvascular flow in the penis increased significantly following SIL administration. The endothelium-dependent skin microvascular vasodilator responses induced by ACh were also significantly enhanced following SIL administration for each of the following parameters: peak values of cutaneous vascular conductance (CVC); increases in CVC; and the area under the curve for ACh-induced vasodilation. ACh-induced microvascular vasodilation in the forearm was not modified by SIL. Finally, the administration of electric current alone did not affect penile microvascular flow. CONCLUSION: LSCI appears to be a promising non-invasive technique for the evaluation of penile microvascular endothelial function. This methodology may be valuable for the evaluation of penile microvascular reactivity among patients with cardiovascular and metabolic diseases and to test the effectiveness of drugs used to treat vasculogenic erectile dysfunction.