The uptake of acidic amino acids by goldfish photoreceptors was investigated by light microscope autoradiography. Isolated retinas were incubated in media containing micromolar amounts of L-[3H]aspartate, L-[3H]glutamate, and D-[3H]aspartate. We have four major observations. (i) Rods accumulate L-[3H]glutamate with a high-affinity transport system; they exhibit a glutamate-to-aspartate selectivity ratio of 30:1. When incubated in 1-10 microM L-[3H]glutamate, rods label more densely than cones. A unit area of rod membrane transports glutamate 30 times better than a unit area of cone membrane. (ii) Red-sensitive and green-sensitive cones show accumulation of L-[3H]aspartate, D-[3H]aspartate, and L-[3H]glutamate, apparently with high affinity, but with little selectivity. Because rods have poor aspartate uptake, red-sensitive and green-sensitive cones may be preferentially labeled with L-[3H]aspartate or D-[3H]aspartate, (iii) Blue-sensitive cones show no uptake of L-[3H]aspartate, D-[3H]aspartate, or L-[3H]glutamate other than that attributable to low-affinity transport. (iv) Various cell types in the goldfish retina can clearly discriminate between glutamate and aspartate, unlike acidic amino acid transport systems described in mammalian brain.