Effects of L-glutamate (Glu), the neurotransmitter released by photoreceptors, on isolated cat bipolar cells were examined. Membrane currents of bipolar cells were recorded by the patch-clamp technique in a conventional whole-cell recording configuration using pipettes containing 1 mM cGMP, which has been known to activate a cationic current sensitive to Glu in ON-type bipolar cells. ON-type bipolar cells (depolarized by light in in situ) and OFF-type bipolar cells (hyperpolarized by light) were identified by their response polarity to Glu. When the whole-cell configuration was established, ON-type bipolar cells showed a steady inward current which was suppressed by Glu, consistent with the response polarity observed in in situ recordings. In contrast, OFF-type cells did not show a steady current during the recordings. However, they responded to Glu with an increase in cationic conductance. Among recorded cells, rod-driven bipolar cells were identified by their immunoreactivity to anti-protein kinase C (PKC-IR) antibody. Examination of PKC-IR revealed that ON-type bipolar cells included both rod- and cone-driven bipolar cells, while OFF-type cells were all cone-driven bipolar cells. The cGMP-activated current observed in ON-type cells was accompanied by a change in the current fluctuation due to the opening and closing of underlying channels. Fluctuation analysis gave a unitary conductance value of 13 pS. In half of the cells examined, maximum open probability reached almost 100%. The cGMP-activated channel in bipolar cells seems novel, fundamentally different from those found in photoreceptor cells or olfactory receptor cells.