Small injections of True Blue (TB) into 4 different segments of the pancreas of the rat resulted in characteristic and different numbers and distributions of labeled cells within the dorsal motor nucleus of the vagus (DMN). Composites of these patterns of labeled cells in the DMN closely matched the distributions previously reported for more extensive injections of retrograde tracers into the pancreas. However, the application of a diffusion barrier (formed with a plastic wound spray) on the outer surface of the stomach and intestines adjacent to the pancreas segment which contained the TB injection depot prevented virtually all of the labeling of DMN cells. Similarly, applying a diffusion barrier directly to the injected pancreas segment itself prevented all or most (mean greater than 99%) of the DMN labeling. In contrast to this effect on DMN label, the barrier reduced more modestly the labeling of celiac ganglion somata after pancreas injections. Several additional control experiments also suggest that the absence of DMN label after the barrier application resulted from interference with tracer diffusion from the injected organ and not from neurotoxic effects. These include the following demonstrations in the presence of the barrier: (1) observation of unimpaired vagally stimulated insulin secretion, (2) uncompromised cell labeling of DMN from other organs treated with TB plus the barrier, and (3) normal hematoxylin and eosin stained pancreas tissue which had received TB injections and barrier application. It was concluded that both the number of parasympathetic preganglionic neurons that project monosynaptically to the pancreas and their distribution in the medulla may have been very significantly overestimated in previous tracer studies.