Glutamate has many important physiological functions including its role as a neurotransmitter in the retina and CNS. We have made the novel observations that retinal pigment epithelial cells underlying and intimately interacting with the retina secrete glutamate and this secretion is significantly affected by iron. In addition, iron increased secretion of glutamate in cultured lens and neuronal cells indicating that this may be a common mechanism for regulation of glutamate production in many cell types. The activity of the iron dependent enzyme, cytosolic aconitase (c-aconitase), is increased by iron. The conversion of citrate to isocitrate by c-aconitase is the first step in a three step process leading to glutamate formation. In the present study, iron increased aconitase activity and this increase was associated with an increase in glutamate secretion. Inhibition of c-aconitase by oxalomalate decreased glutamate secretion and completely inhibited the iron induced increase in glutamate secretion. Derangements in both glutamate secretion and iron metabolism have been noted in neurological diseases and in retinal degeneration. The results of the present investigation are the first to provide a functional link between these two physiologically important substances by demonstrating a significant role for iron in regulation of glutamate production and secretion in mammalian cells resulting from iron regulation of aconitase activity. Glutamatergic systems are found in many non-neuronal tissues. In the present study we provide the first evidence that in addition to secreting glutamate, retinal pigment epithelial cells express the vesicular glutamate transporter, VGLUT1, and that regulated vesicular release of glutamate from these cells can be inhibited by riluzole.