OBJECTIVE To determine flash and background colors that best isolate the photopic negative response (PhNR) and maximize its amplitude in the primate ERG. METHODS Photopic full-field flash ERGs were recorded from anesthetized macaque monkeys before and after pharmacologic blockade of Na(+)-dependent spiking activity with tetrodotoxin (TTX, 1 to 2 muM, n = 3), blockade of ionotropic glutamatergic transmission with cis-2,3 piperidine dicarboxylic acid (PDA, 3.3-3.8 mM, n = 3) or laser-induced monocular experimental glaucoma (n = 6), and from six normal human subjects. Photopically matched colored flashes of increasing stimulus strengths were presented on scotopically matched blue, white, or yellow backgrounds of 100 scot cd/m(2) using an LED-based stimulator. RESULTS PhNRs that could be eliminated by TTX or severe experimental glaucoma were present in responses to brief (<5 ms) and long-duration (200 ms) stimuli of all color combinations. In normal monkey and human eyes for brief low-energy flashes, PhNR amplitudes were highest for red flashes on blue backgrounds and blue flashes on yellow backgrounds. For high-energy flashes, amplitudes were more similar for all color combinations. For long-duration stimuli, the PhNR(on) at light onset in monkeys was larger for red and blue stimuli, regardless of background color, than for spectrally broader flashes, except for stimuli >17.7 cd/m(2) when PhNR(on)s were all of similar amplitude. For red flashes, eliminating the PhNR(on) pharmacologically or by glaucoma removed the slowly recovering negative wave that normally followed the transient b-wave and elevated the whole ON response close to the level of the b-wave peak. However, for white, blue, and green flashes, a lower-amplitude plateau that could be removed by PDA remained. CONCLUSIONS For weak to moderate flash strengths, the best stimulus for maximizing PhNR amplitude is one that primarily stimulates one cone type, on a background with minimal adaptive effect on cones. For stronger stimuli, differences in amplitude are smaller. For long-duration stimuli, red best isolates the PhNR(on) because it minimizes the overlapping lower-level plateau that originates from the activity of second-order hyperpolarizing retinal neurons.