Nerve growth factor (NGF) production in the cultured rat iris was examined using immunohistochemistry and bioassay of irides and conditioned media. NGF immunoreactivity increased steadily with days in culture so that the intensity of staining was maximal after 6 days of culture. The localization was shown to be sensitive to the presence of cross-linking fixatives such as formaldehyde and glutaraldehyde and this effect was only partially alleviated by the use of very high concentrations of antibodies. NGF immunoreactivity was localized in Schwann cells and possibly nerve axons, but with no antigen detectable in smooth muscle fibres. Media conditioned over irides initially supported a high percentage of dissociated sympathetic neurons, but the number supported decreased with time in culture until day 4. Moreover, the use of antibodies to NGF allowed the detection of at least two types of neuronotropic activity, NGF accounting for at least 94% of the total trophic activity present after 4 days of culture. These findings provide support for the proposal that Schwann cells produce NGF and question the accepted hypothesis that the molecule is produced by smooth muscle fibres as a peripheral maintenance factor for sympathetic and sensory nerves. The results also suggest that two survival factors may be involved in the regulation of sympathetic function.