Substantial evidence suggests that vagal afferent functions are modulated by agonists acting on afferent terminals in the solitary nucleus (NST). Actions of these agonists are implied through intracellular recordings from cultured nodose ganglion cells or second order NST neurons. While high-quality data have been obtained using these methods, techniques in which physiological measurements can be made directly on the afferent terminal fields, in situ, in the NST, would eliminate several potential interpretive problems inherent in these less direct approaches. This paper describes methods developed to directly measure changes in presynaptic cytoplasmic calcium in vagal afferents using time-lapse laser confocal microscopy on the in vitro brainstem slice. Calcium green dextran (CG) transported from the nodose ganglion clearly demonstrates vagal afferent fibers ramifying throughout the NST in the in vitro brainstem slice. CG-labeled vagal afferents can be repeatedly activated by focal electrical stimulation, by agonists acting on presynaptic ligand-gated ion channels, and by molecules that are presumed to act directly on vagal afferents based on previous physiological and immunocytochemical studies. Image and preparation stability are a challenge to the success of the experiment; however, methods described here should assist direct studies of transduction events within other afferent terminal fields in the CNS.