The developmental influence of sympathetic innervation on parasympathetic nerve density was investigated in the tarsal smooth muscle of the rat. Specificity of acetylcholinesterase staining as a marker for parasympathetic innervation was first determined by acute selective denervations. Excision of the ipsilateral superior cervical ganglion caused a 39% reduction in the density of acetylcholinesterase-positive nerves seven days later, indicating that sympathetic nerves contribute to cholinesterase-positive tarsal muscle innervation. Excision of the pterygopalatine ganglion concurrent with superior cervical ganglionectomy caused a virtually complete disappearance of acetylcholinesterase-positive innervation within seven days, indicating that non-sympathetic cholinesterase-positive fibers derive from the pterygopalatine ganglion and are presumed to be parasympathetic. Analysis of the control population indicated that parasympathetic nerve density did not vary significantly between males and females, between the superior and inferior muscles, or in rats studied at four and 12 months of age. The influence of sympathetic innervation on parasympathetic nerve density during postnatal development was examined by conducting surgical sympathectomies on postnatal day 5 and quantifying acetylcholinesterase-positive nerve density at four months of age. Neonatal sympathectomy caused a 46% reduction in cholinesterase-positive nerve density beyond that which occurred in acutely sympathectomized adult controls. It is concluded that sympathetic innervation is required for developing parasympathetic nerves to attain their normal density within the rat tarsal muscle. This finding is consistent with the idea that sympathetic nerves can exert positive effects on parasympathetic nerve outgrowth during development.