Neuropeptide Y is stored in sympathetic nerve terminals throughout the heart and has direct and indirect effects on cardiac function. Although neuropeptide Y has been shown to be released upon intense (16-30 Hz) cardiac sympathetic stimulation, we sought to determine whether effective quantities of neuropeptide Y were released from cardiac sympathetic neurons under more natural conditions. We recorded arterial pressure and cardiac cycle length in 29 anesthetized dogs. We assessed neuropeptide Y release by measuring the attenuation of vagally induced increases in cardiac cycle length (10 seconds every 2 minutes) after trams of sympathetic stimulation. We examined the effect of constant-frequency sympathetic stimulation (frequencies of 2, 5,10, and 15 Hz, applied for train durations of 1,3, and 5 minutes) on vagally induced chronotropic responses. We also determined the effect of varying the pattern of sympathetic stimulation. Both the magnitude and duration of the inhibition of the vagal effects on cardiac cycle length were augmented significantly by increases in the frequency or duration of sympathetic stimulation. In contrast, the inhibition of the vagally induced chronotropic responses was not significantly affected by changes in the pattern of sympathetic stimulation. We also characterized the role of adrenergic receptors. Phentolamine significantly increased the sympathetically mediated inhibition of the vagal effects on cardiac cycle length, but propranolol had no effect. We concluded that neuropeptide Y release from cardiac sympathetic neurons 1) depends on the frequency and duration, but not on the pattern, of sympathetic stimulation; 2) is evoked even at physiological frequencies (2-5 Hz) of sympathetic activity; and 3) is enhanced by a-adrenergic-receptor blockade, but is unaffected by β-adrenergic- receptor blockade.