Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably alpha-smooth muscle actin (alpha-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at -20 degrees C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express alpha-SMA. Junctional pericytes were more frequently alpha-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (alpha-SMA-siRNA) suppressed alpha-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of alpha-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express alpha-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling.