Inhibition of norepinephrine release by opioid agonists is inversely related to the stimulation train length. The possible interaction between activation of prejunctional alpha-2 adrenergic receptors and the release-inhibiting opioid receptors as well as the effect of changes in Ca++ entry into adrenergic varicosities during repetitive stimulation were investigated by recording vasoconstriction of the rabbit ear artery perfused in vitro. Neither the activation of the alpha-2 adrenoceptor-mediated negative feedback by clonidine nor its inhibition by yohimbine altered the neuroinhibitory potency for dynorphin 1-13 or Met-enkephalin at any stimulus train length. Experimental conditions known to increase the entry of calcium into the varicosities mimicked the effect of the increase in the stimulation train length on the modulation of norepinephrine release by opioids. Increasing the extracellular calcium concentration (from 1.6-5 or 8 mM) diminished the inhibitory effect of opioids and tended to abolish the dependence on stimulation train length. Conversely, lowering the calcium concentration (from 1.6-1 mM) increased the inhibition by opioids and enhanced the dependence on train length. These results do not suggest a direct interaction between activation of opioid receptors and alpha-2 adrenoceptors. Rather, they indicate the role of opioid receptor activation in a primary modulation of Ca++ influx which becomes masked by the high levels of axoplasmic calcium which are achieved during a continued stimulation train.