1. Single-unit recording and iontophoretic techniques were used to test the effects of serotonin (5-HT) on the responses of neurons in the superficial layers (the stratum griseum superficiale and stratum opticum) of the hamster's superior colliculus (SC). 2. Iontophoresis of 5-HT produced a visual response suppression of 40% or greater in 78.1% (n = 50) of 64 neurons tested. 5-HT did not augment the visual responses of any of the cells tested. The average response suppression was 75.3 +/- 21.2% (mean +/- S.D.). 3. Iontophoresis of 5-HT had significantly different effects on activation of SC cells by optic chiasm (OX) and visual cortical (CTX) stimulation. Application of 5-HT suppressed the OX-evoked responses of 96.9% (n = 31) of the 32 SC cells tested by at least 40%, and the average response suppression for all 32 neurons tested was 87.1 +/- 22.5%. Application of 5-HT suppressed the responses of only 35.7% (n = 10) of the 28 cells tested with CTX stimulation by at least 40%. The average response suppression for all 28 cells was 35.3 +/- 38.8%. 4. The effects of 5-HT on the glutamate-evoked responses of SC cells that were synaptically "isolated" by concurrent application of Mg2+ were also evaluated. Application of 5-HT produced a response suppression > or = 40% in 29.7% (n = 19) of the 64 neurons tested under these conditions. The average response suppression for all of the cells tested was 28.4 +/- 35.7%. This effect of 5-HT was significantly weaker than that on visually evoked responses of these neurons. 5. The present results demonstrate that 5-HT markedly depresses the visual responses of most superficial layer SC neurons. They suggest further that much of this effect is mediated by presynaptic inhibition of retinotectal transmission.