A system of indoleamine-accumulating neurons exists in the retina of several species, but the exact transmitter of these neurons is not known. 5-Hydroxytryptamine (5-HT) is a likely candidate, but there are conflicting reports on its amount in the retina. We have determined the amounts of 5-HT with two sensitive, specific, and independent methods. Because the content was found to be lower than what was expected for a neurotransmitter, the rate-limiting enzyme in the 5-HT synthesis was also estimated, as was the effect of 5-HT on cyclic AMP content of the retina. In the rabbit the 5-HT content was found to be 25 to 35 ng/gm wet weight, with not difference between light- and dark-adapted animals. There was no detectable activity of the rate-limiting enzyme tryptophan hydroxylase. It was not possible to raise the 5-HT concentration by treating the rabbits with a monoamine oxidase inhibitor and L-tryptophan. 5-HT induced no change in the cyclic AMP content of the rabbit retina. In both newly hatched and older chicken retina, the 5-HT concentration was higher than in rabbit. The 5-HT concentrations in all retinas were lower than would be expected for a monoamine neurotransmitter, and these results argue against 5-HT being the neurotransmitter of the indoleamine-accumulating neurons.