Introduction: The sympathetic nervous system can modulate arteriolar tone through release of adenosine triphosphate and norepinephrine, which bind to purinergic and adrenergic receptors (ARs), respectively. The expression pattern of these receptors, as well as the composition of neurotransmitters released from perivascular nerves (PVNs), can vary both in organ systems within and across species, such as mice and rats. Objective: This study explores the function of α<sub>1A</sub> subtypes in mouse and rat third-order mesenteric arteries and investigates PVN-mediated vasoconstriction to identify which neurotransmitters are released from sympathetic PVNs. Methods: Third-order mesenteric arteries from male C57BL/6J mice and Wistar rats were isolated and mounted on a wire myograph for functional assessment. Arteries were exposed to phenylephrine (PE) and then incubated with either α<sub>1A</sub> antagonist RS100329 (RS) or α<sub>1D</sub> antagonist BMY7378, before reexposure to PE. Electrical field stimulation was performed by passing current through platinum electrodes positioned adjacent to arteries in the absence and presence of a nonspecific alpha AR blocker phentolamine and/or P2X<sub>1</sub>-specific purinergic receptor blocker NF449. Results: Inhibition of α<sub>1</sub> ARs by RS revealed that PE-induced vasoconstriction is primarily mediated through α<sub>1A</sub> and that the contribution of the α<sub>1A</sub> AR is greater in rats than in mice. In the mouse model, sympathetic nerve-mediated vasoconstriction is mediated by both ARs and purinergic receptors, whereas in rats, vasoconstriction appeared to only be mediated by ARs and a nonpurinergic neurotransmitter. Further, neither model demonstrated that α<sub>1D</sub> ARs play a significant role in PE-mediated vasoconstriction. Conclusions: The mesenteric arteries of male C57BL/6J mice and Wistar rats have subtle differences in the signaling mechanisms used to mediate vasoconstriction. As signaling pathways in humans under physiological and pathophysiological conditions become better defined, the current study may inform animal model selection for preclinical studies.