The malignant transformation of congenital nevocellular nevi, both large and small, is controversial and presents problems in management. The size of the lesion is taken to indicate potential malignant transformation, but this is an arbitrary scale. A more reliable biological indicator is needed to help predict the lesions at risk. Following the localization of neuron-specific enolase to most cells of the diffuse neuroendocrine system and their neoplasms (including benign and malignant melanocytic lesions), it has been suggested that its level is related to tumor activity. In a prospective trial, the presence of neuron-specific enolase immunoreactivity, its concentration, and gene expression in nevus cells were studied in 31 congenital melanocytic nevi of various sizes (1.5 cm to bathing trunk) using immunocytochemistry, biochemical assay, and in situ hybridization. Twenty-five of the 31 congenital nevi were immunoreeactive to neuron-specific enolase antiserum, with stronger immunostaining in the larger lesions. There is an apparent linear relationship between the size of the nevi and the level of neuron-specific enolase (expressed as nanograms per milligram protein). Neuron-specific enolase mRNA was highly expressed in most of the large congenital nevi (greater than 15 cm in diameter), as revealed by autoradiography following in situ hybridization. Our results show that neuron-specific enolase and its mRNA are expressed to a greater extent in large congenital nevi compared with the smaller lesions. This might prove to be a useful indicator of those lesions at risk of malignant transformation.