The mechanisms of autonomic imbalance and subsequent cardiovascular manifestations in HIV-1-infected patients are poorly understood. We report here that HIV-1 trans-activator of transcription (Tat, fragment 1-86) produced a concentration-dependent increase in cytosolic Ca²⁺ in cardiac-projecting parasympathetic neurons of nucleus ambiguus retrogradely labeled with rhodamine. Using stores-specific pharmacological agents, we identified several mechanisms of the Tat-induced Ca²⁺ elevation: (1) lysosomal Ca²⁺ mobilization; (2) Ca²⁺ release via inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools; and (3) Ca²⁺ influx via transient receptor potential vanilloid type 2 (TRPV₂) of nonselective cation channels. Activation of TRPV₂, nonselective cation channels, induced a robust and prolonged neuronal membrane depolarization, thus triggering an additional P/Q-mediated Ca²⁺ entry (4). In vivo microinjection studies indicate a dose-dependent, prolonged bradycardic effect of Tat administration into the nucleus ambiguus of conscious rats, in which neuronal TRPV₂ played a major role. Our results support previous studies indicating that Tat promotes bradycardia and consequently may be involved in the QT interval prolongation reported in HIV-infected patients. In the context of an overall HIV-dependent autonomic dysfunction, these Tat-mediated mechanisms may account for the higher prevalence of sudden cardiac death in HIV-1-infected patients as compared with general population with similar risk factors. Our results may be particularly relevant in view of the recent findings that significant Tat levels can still be identified in the cerebrospinal fluid of HIV-infected patients with viral load suppression due to efficient antiretroviral therapy.