Previous studies showed that the cardiac response of the baroreceptor reflex (bradycardia) is inhibited during the defense reaction evoked by direct electrical or chemical stimulation of the periaqueductal gray (dPAG) in the rat. Whether central serotonin and nucleus tractus solitarius (NTS) 5-HT₃ receptors might participate in this inhibition was investigated in urethane anaesthetized-, atenolol-pretreated, rats. Our results showed that both electrical and chemical stimulation of the dPAG produced a drastic reduction of the cardiovagal component of the baroreceptor reflex triggered by either intravenous administration of phenylephrine or aortic nerve stimulation. This inhibitory effect of dPAG stimulation on the baroreflex bradycardia was not observed in rats that had been pretreated with p-chlorophenylalanine (PCPA, 300 mg/kg i.p. daily for 3 days) to inhibit serotonin synthesis. Subsequent 5-hydroxytryptophan administration (60 mg/kg i.p.), to restore serotonin synthesis, allowed the inhibitory effect of dPAG stimulation on both aortic- and phenylephrine-induced cardiac reflex responses to be recovered in PCPA-pretreated rats. On the other hand, in non pretreated rats, the inhibitory effect of dPAG stimulation on the cardiac baroreflex response could be markedly reduced by prior intra-NTS microinjection of granisetron, a 5-HT₃ receptor antagonist, or bicuculline, a GABA_A receptor antagonist. These results show that serotonin plays a key role in the dPAG stimulation-induced inhibition of the cardiovagal baroreceptor reflex response. Moreover, they support the idea that 5-HT₃ and GABA_A receptors in the NTS contribute downstream to the inhibition of the baroreflex response caused by dPAG stimulation.