<jats:title>ABSTRACT</jats:title><jats:p><jats:bold>Objective</jats:bold>: The goal of the present study was to develop a method to isolate viable arteriolar muscle cells from single cremasteric arterioles, which retain the contractile and electrophysiological phenotype of the donor microvessels.</jats:p><jats:p><jats:bold>Methods</jats:bold>: Arterioles were hand‐dissected from rat and hamster cremaster muscles and dissociated by incubation in papain and dithioerythritol for 35 min followed by incubation in collagenase, elastase, and soybean trypsin inhibitor for 10 to 25 min in solutions containing 100 μM Ca<jats:sup>2+</jats:sup>, 10 μM sodium nitroprusside, and 1 mg/ml albumin at 37°C.</jats:p><jats:p><jats:bold>Results</jats:bold>: Populations of single smooth muscle cells enzymatically isolated from cremasteric arterioles showed elongated fusiform morphology and intact plasmalemmal membranes as indicated by retention of calcein, by exclusion of ethidium ho‐modimer‐1, and by high membrane resistances (11 ± 0.8 Cω, <jats:italic>n</jats:italic> = 36 for rat cells: 8 ± 0.6 Cω, <jats:italic>n</jats:italic> = 21 for hamster cells: <jats:italic>p</jats:italic> < 0.05). Muscle cells contracted in a concentration‐dependent fashion in response to pipette application of norepinephrine (10 nM‐100 μ). Cell shortening in response to 1 μM norepinephrine was inhibited by 10 μM phentolamine, 1 μM sodium nitroprusside, and 1 μM nifedipine or nominally Ca<jats:sup>2+</jats:sup>‐free media. Resting membrane potential recorded in patch‐clamped cells by perforated patch methods was −48 ± 1 mV (<jats:italic>n</jats:italic> = 47) for rat cells and −44 ± 2.8 mV (<jats:italic>n</jats:italic> = 14) for hamster cells (<jats:italic>p</jats:italic> > 0.05). Families of voltage‐dependent K<jats:sup>+</jats:sup> currents were observed during stepwise depolarizing pulses from −60 mV to more positive potentials. Blockers of voltage‐gated and ATP‐sensitive K<jats:sup>+</jats:sup> channels (4‐aminopyridine [3 mM] and glibenclamide [1 μM], respectively) inhibited membrane K<jats:sup>+</jats:sup> conductance, increased membrane resistance, and depolarized cells by 20 ± 4 mV (<jats:italic>n</jats:italic> = 8) and 14 ± 3 mV (<jats:italic>n</jats:italic> − 6), respectively.</jats:p><jats:p><jats:bold>Conclusions</jats:bold>: The present method permits isolation of smooth muscle cells from a single cremasteric arteriole. These cells seem to retain the contractile phenotype, α‐adrenergic signaling cascade, membrane potential, and K<jats:sup>+</jats:sup> conductances described for the donor arteriole. Correlating the functional and electrophysiological properties of these smooth muscle cells to <jats:italic>in situ</jats:italic> and <jats:italic>in vitro</jats:italic> studies of their donor arterioles should provide a useful extension for understanding the physiology, patho‐physiology, biophysics, and cell biology of the microcirculation in skeletal muscle.</jats:p>