(1) Rat cortical neurons grown in dissociated cell culture exhibit IPSPs which appear to be generated by an increase in membrane conductance to chloride. (2) The neurons are all sensitive to GABA in micromolar concentrations and GABA mimics the inhibitory transmitter. (3) The neurons are much less sensitive to glycine and insensitive to taurine. (4) Bicuculline and strychnine both block essentially all IPSPs and at the same concentrations block GABA effects. (5) It is concluded that GABA is the main, or only, inhibitory transmitter utilized by the cortical neurons in vitro. The relevance of this conclusion to in situ transmitter identification is discussed.