A cross-interaction between GABA- and glycine-evoked currents was found when the two transmitters were applied in sequence to cultured embryonic rat hippocampal neurons. Whole-cell GABA-current was inhibited by a previous glycine-current flowing in the same direction (inward or outward), and potentiated by a current with opposite polarity. The same effect was caused by GABA on glycine-current. Repeated applications of GABA (glycine) elicited currents of decreasing or increasing amplitude, according to a similar pattern. Transmitter interaction was independent of external Ca2+ and of all the metabolic pathways tested, but it was blocked by specific receptor antagonists, bicuculline and strychnine. The extent of both inhibition and potentiation correlated with the amount of charge flowing through the membrane during the conditioning transmitter application, indicating that cross-modulation depends on shifts of Cl- reversal potential. This finding has both functional and methodological implications, as it suggests a new mechanism of transmitter interaction in the brain, and also that patch-clamp pipettes cannot adequately perfuse cell interior.