Evidence to support a neurotransmitter role for 5-hydroxytryptamine in chick retain is presented. Putative 5-hydroxytryptophan and 5- hydroxytryptamine extracted from chick retina have been characterized chromatographically and electrochemically and have been found to behave like the authentic compounds. Retinal tryptophan hydroxylase activity could not be demonstrated in vitro, but chick retina was able to synthesize 5-[3H]hydroxytryptamine from [3H]tryptophan in vivo. Retinal 5-hydroxytryptamine levels were reduced by local injection of alpha- fluoromethyldopa, an inhibitor of aromatic L-amino acid decarboxylase, or reserpine, indicating that his amine is both synthesized and stored in this tissue. The retinae of light-adapted chicks have higher levels of 5-hydroxytryptamine and its major metabolite, 5-hydroxyindoleacetic acid, than dark-adapted animals. The turnover of 5-hydroxytryptamine appears to be greater in the light after inhibition of synthesis. Conversely, the rate of synthesis of 5-hydroxytryptamine appears to be greater in the dark. The possible interaction of dopaminergic neurons and the 5-hydroxytryptamine-containing cells and the effect of alpha- fluoromethyldopa on this relationship are discussed. The data lend support to the notion that 5-hydroxytryptamine in chick retina may have a neurotransmitter function.