Innervation plays an important role in the maintenance of corneal structure and function. Neuropeptides contained in corneal nerves may be involved in these effects. This study was aimed to determine the influence of trigeminal and sympathetic neurons and of neuropeptides on proliferative activity of corneal epithelial cells in vitro. Corneal epithelial cells from adult rabbits were cultured alone or cocultured either with sensory neurons from the trigeminal ganglion or sympathetic cells from the superior cervical ganglion of newborn rabbits. Neurons and corneal epithelial cells shared the culture medium but lacked physical contact. After 2 or 7 days of coculture, or after treatment with substance P (SP) and/or calcitonin gene-related peptide (CGRP), the number of corneal cells was determined with a haemocytometer, and the incorporation of 3H-thymidine into corneal epithelial cells analysed by scintillation counting. Cell number and 3H-thymidine incorporation increased 3 and 2.5 times respectively (P < 0.01; P < 0.05), after 2 days of coculture of corneal epithelial cells with trigeminal neurons but this increase was reversed after 7 days of coculture. Sympathetic neurons had a stimulating effect on corneal epithelial cell proliferation that was apparent after 7 days of coculture (P < 0.01). A single administration of SP (10 microM) produced an increase in 3H-thymidine incorporation after 1 hr and an enhanced number of corneal epithelial cells 24 hr later. The same effect could be obtained by repeated administration of lower concentrations of SP (0.1 microM, six times over a 24 hr period). CGRP (1 microM) inhibited mitotic activity of corneal cells 12 hr after treatment. The effect could be reversed by simultaneous administration of SP (0.1 microM). These results indicate that trigeminal and sympathetic neurons modulate proliferation and perhaps differentiation of corneal epithelium cells in vitro. A stimulating effect was also obtained with SP while mitotic activity was inhibited by CGRP, thus suggesting that peptides contained in sensory nerve terminals may contribute to neural influences on epithelial cell proliferation.