Postganglionic sympathetic neurons and brain noradrenergic neurons use alpha(2A)- and alpha(2C)-adrenoceptors as presynaptic autoreceptors. The present experiments were carried out in order to see whether they possess presynaptic alpha(2B)-autoreceptors as well. Pieces of atria, vasa deferentia, the occipito-parietal cortex and the hippocampus were prepared from either wildtype (WT) mice or mice in which both the alpha(2A)- and the alpha(2C)-adrenoceptor gene had been disrupted (alpha(2AC)KO). The pieces were incubated with (3)H-noradrenaline and then superfused and stimulated electrically. In a first series of experiments, single pulses or brief, autoinhibition-poor pulse trains were used for stimulation. The alpha(2)-adrenoceptor agonist UK 14,304 (brimonidine) reduced the evoked overflow of tritium in all four tissues from WT mice but did not change it in any tissue from alpha(2AC)KO mice. A different pattern was obtained with medetomidine as alpha(2 )agonist. Like UK 14,304, medetomidine reduced the evoked overflow of tritium in all four tissues from WT mice and did not affect overflow in brain slices from alpha(2AC)KO mice; however, in contrast to UK 14,304, medetomidine reduced evoked overflow also in atrial and vas deferens pieces from alpha(2AC)KO mice, although with a lower maximum and potency than in WT preparations. The alpha-adrenoceptor antagonists rauwolscine, phentolamine, prazosin, spiroxatrine and WB 4101 shifted the concentration-response curve of medetomidine in alpha(2AC)KO atria and vasa deferentia to the right. The pK(d) values of the five antagonists against medetomidine in alpha(2AC)KO atria and vasa deferentia correlated with pK(d) values at prototypical alpha(2B) radioligand binding sites but not at alpha(2A) or alpha(2C) binding sites. In a second series of experiments, autoinhibition-rich pulse trains were used for stimulation. Under these conditions, rauwolscine and phentolamine increased the evoked overflow of tritium from alpha(2AC)KO atrial and vas deferens pieces but not from alpha(2AC)KO brain slices. The increase was smaller (by 40% in atria and by 70% in the vas deferens) than previously observed in WT preparations (by 200-400%). In a last series of experiments, mRNA for the alpha(2B)-adrenoceptor was demonstrated by RT-PCR in thoracolumbar sympathetic ganglia from WT, alpha(2A)KO, alpha(2C)KO and alpha(2AC)KO mice but not from alpha(2B)KO mice. The results show that brain noradrenergic neurons express only alpha(2A)- and alpha(2C)-adrenoceptors as autoreceptors. Postganglionic sympathetic neurons, however, can express alpha(2B)-adrenoceptors as presynaptic autoreceptors as well. The alpha(2B)-autoreceptors are activated by medetomidine but not by UK 14,304. They are also activated by previously released noradrenaline. The two-alpha(2)-autoreceptor hypothesis has to be replaced by a three-autoreceptor hypothesis for postganglionic sympathetic neurons.