The regional distributions of acetylcholine (ACh) and choline (Ch) in the guinea pig heart were investigated with a pyrolysis-mass fragmentography technique. Using ACh as a marker for cholinergic neurons, we have described a pattern of parasympathetic innervation in the guinea pig heart. This distribution is very similar to that suggested by studies using several different cholinergic indicators in various species. Atrial areas receive richer parasympathetic innervation than ventricular areas, with the right portions receiving more than the left. The nodal areas were the most abundantly innervated regions examined. Ch content is not a good indicator for cholinergic innervation as the regional distribution of ACh and Ch throughout the guinea pig heart are not strongly associated.