Cardiac vagal tone is a strong predictor of health, but its central origins are unknown. The rat working heart-brainstem preparation (WHBP) shows strong cardiac vagal tone and pronounced respiratory sinus arrhythmia. In this preparation, recordings from the cut left cardiac vagal branch showed efferent activity that peaked in postinspiration, ∼0.5 s before the cyclic minimum in heart rate. We hypothesised that respiratory modulation of cardiac vagal tone and heart rate is intrinsically linked to the generation of postinspiration. Neurons in the pontine Kölliker-Fuse nucleus (KF) were inhibited with bilateral microinjections of isoguvacine (50-70 nl, 10 mM) to remove the postinspiratory phase of respiration. This also abolished the postinspiratory peak of cardiac vagal discharge (and cyclical heart rate modulation), but a substantial level of activity remained. In separate preparations with intact cardiac vagal branches but sympathetically denervated by thoracic spinal pithing, cardiac chronotropic vagal tone was quantified by heart rate compared with its final level after systemic atropine (0.5 μM). Bilateral KF inhibition removed 88% of the cyclical fluctuation in heart rate but, on average, only 52% of the chronotropic vagal tone. Substantial chronotropic vagal tone also remained after transection of the brainstem through the caudal pons. Subsequent bilateral isoguvacine injections into the nucleus of the solitary tract (NTS) further reduced vagal tone: remaining sources were untraced. We conclude that cardiac vagal tone depends on neurons in at least three sites of the pontomedullary brainstem, and much of it arises independently of respiratory sinus arrhythmia.