Retinal bipolar neurons release the excitatory transmitter, glutamate. However, certain bipolar cells contain GABA, raising the question whether a neuron might release both transmitters and, if so, what function might the inhibitory transmitter play in a particular circuit? Here we identify a subset of cone bipolar cells in cat retina that contain glutamate, plus its vesicular transporter (VGLUT1), and GABA, plus its synthetic enzyme (GAD65) and its vesicular transporter (VGAT). These cells are negative for a marker of ON bipolar cells and restrict their axons to the OFF strata of the inner synaptic layer. They do not colocalize with the neurokinin 3 receptor that stains a type (or two) of OFF bipolar cells. By “targeted injection,” we identified two types of OFF bipolar cell with the machinery to make and package both transmitters. One of these types costratifies with a dopamine plexus. J. Comp. Neurol. 478:207–218, 2004. © 2004 Wiley-Liss, Inc.