The purpose of this study is to determine whether differences in membrane potential and/or intracellular Ca2+ increments are implicated in a programmed release of amino acid neurotransmitters (aspartate, glutamate, glycine and GABA) in cortical neurons in culture. According to our results, it is possible to assume that difference in membrane potential is not the only signal which starts the amino acid neurotransmitter release, but there are other necessary conditions at the start of this amino acid release. One of these conditions could be the increment in intracellular Ca2+, but our results indicate that, in cortical neurons in culture, the total intracellular Ca2+ increments are not important on release levels, but are the stimulating agent which produces this intracellular Ca2+ increment. From these results we may infer: (1) that in rat cortical neurons there are neurons which contain and release glutamate, aspartate, glycine and GABA, (2) that in cortical neurons the 36.6 +/- 5.8% of the neurons are GABA-ergic, (3) that the membrane potential and the total intracellular calcium are not only responsible for the release of these amino acids but also the depolarizing agent which plays an important role in this release, and (4) that glutamate and aspartate and glutamate and GABA are localized in different vesicular pools or in different cell neurons.