The ERG b-wave is widely believed to reflect mainly light-induced activity of on-center bipolar cells and Müller cells. Third-order retinal neurons are thought to contribute negligibly to generation of the b-wave. Here we show that pharmacological agents which affect predominantly third-order neurons alter significantly both the kinetics and amplitude of the b-wave. Our results support the notion that changes in the amplitude and kinetics of light-induced membrane depolarization in third-order neurons produce similar changes in the amplitude and kinetics of the b-wave. We conclude that activity in third-order neurons makes a significant contribution to b-wave generation. Our results also provide evidence that spiking activity of third-order neurons truncates the a-wave by accelerating the onset of the b-wave.