We tested the hypothesis that a light-evoked increase in [K+]o produces a depolarization of the Müller cell membrane, which in turn generates the electroretinogram b-wave current. Using Bufo marinus isolated retinas and K+-selective microelectrodes, we recorded two distinct light-evoked increases in extracellular K+ concentration: one in the inner plexiform layer and the other near the outer plexiform layer; the "distal" K+ increase was found over only 10-μm depth and had a maximum amplitude of 0.3 mM. We also recorded the electroretinogram and the light-evoked responses of rods and Müller cells. After correction for the response time of the K+-selective microelectrode, the waveforms of all three of these responses were almost exactly as predicted by an earlier computer simulation of the K+/Müller cell hypothesis of the b-wave by Newman and Odette [Newman, E. A. & Odette, L. L. (1984) J. Neurophysiol. 51, 164-182]. The distal K+ increase and the b-wave varied in a similar manner as a function of stimulus irradiance. Superfusion with 0.2 mM Ba2+ attenuated both the Müller cell depolarization and the b-wave by ≈65% but had no significant effect upon the distal K+ increase. Because Ba2+ reduces K+ conductance of Müller cells, these results are very strong support of the K+/Müller cell hypothesis of the origin of the electroretinogram b-wave; the light-evoked increase in extracellular potassium concentration still is present during superfusion with Ba2+, but the K+-evoked Müller cell depolarization and the b-wave are decreased in amplitude because Müller cell K+ conductance is reduced.