BACKGROUND: We had previously demonstrated that guanfacine, an alpha2a-adrenergic agonist, attenuated the effect of stress on smoking-lapse behavior in regular daily smokers. Heart rate variability (HRV), a measure of vagal activity, may be a potential mechanism underlying the relationship between stress, smoking, and relapse. METHODS: We examined whether guanfacine (0 mg/day vs. 3 mg/day; n = 26) altered changes in high-frequency heart rate variability (HF-HRV) following stress and ad-lib smoking using a validated laboratory analogue of smoking-lapse behavior. All participants completed a parent study evaluating the effects of guanfacine on stress-precipitated smoking. Each subject completed two laboratory sessions assessing the effects of guanfacine on HF-HRV following stress imagery (vs. neutral imagery; order counterbalanced) and smoking. RESULTS: Results demonstrated that guanfacine did not increase tonic levels of HF-HRV relative to placebo. Following the stress versus neutral imagery manipulation (prior to ad-lib smoking), there were no significant changes in HF-HRV in the placebo group. In contrast, guanfacine increased phasic HF-HRV following stress imagery and decreased HF-HRV following neutral imagery. Ad libitum smoking following both the stress and neutral conditions decreased HF-HRV in the placebo group across both imagery conditions. In contrast, guanfacine attenuated stress- and smoking-related decreases in phasic HF-HRV relative to the neutral imagery condition. CONCLUSIONS: This is the first demonstration that a noradrenergic target altered dynamic changes in HF-HRV in response to stress and smoking, suggesting that guanfacine alters HF-HRV response to stress. Findings support current theories which suggest that phasic changes in HRV are an important marker of the stress response.