A great deal of knowledge exists regarding neural control of myocardial function in the rat. Most of the studies addressing this issue were conducted either under general anaesthesia or in isolated hearts in vitro. Our principal aim was to provide a detailed quantitative description of mechanisms controlling cardiac contractility in the rat, in an anaesthetic-free preparation with a preserved functional brainstem. Furthermore, while vagally mediated negative inotropy is a well-known phenomenon, at present there is no direct evidence for its presence in the rat; we searched for such evidence. To this end, in the arterially perfused working heart-brainstem preparation of the rat, we measured left ventricular pressure (LVP) and computed its first derivative (LVdP/dt). We made the following new observations. (i) Zatebradine (cardiac sodium pacemaker current blocker) caused a bradycardia associated with increases in LVP and LVdP/dt; the latter effect was via a frequency-dependent mechanism. (ii) We confirmed that in the rat, the force-frequency relationship (dependence of contractility on heart rate) is positive over a low range of heart rates, and negative and linear at physiological levels of heart rate, and provided its quantitative description. (iii) The increase in systemic pressure caused a rise in contractility, and vagal blockade or destruction of the central nervous system did not alter this inotropic effect, suggesting that it was mediated by intrinsic cardiac mechanisms. (iv) Vagal stimulation caused complex polyphasic changes in LVdP/dt and LVP in unpaced preparations; during pacing, it caused slowly developing falls in LVdP/dt that could be prevented by atropine. We conclude that control of ventricular contractility in the rat heart differs from that in other mammals not only by its negative frequency dependence, but also in the potent influence of aortic pressure on LVdP/dt. At the level of autonomic neural control, our newly found, vagally mediated negative inotropic effect adds to the accumulating body of data regarding both the presence and the functional importance of parasympathetic innervation of the ventricular myocardium.