The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is a large channel permeable to anions, cations, ATP, and other metabolites. VDAC was purified from sheep brain synaptosomes or rat liver mitochondria using a reactive red-agarose column, in addition to the hydroxyapatitate column. The red-agarose column allowed further purification (over 98%), concentration of the protein over ten-fold, decreasing Triton X-100 concentration, and/or replacing Triton X-100 with other detergents, such as Nonidet P-40 or octylglucoside. This purified VDAC reconstituted into planar-lipid bilayer, had a unitary maximal conductance of 3.7 +/- 0.1 nS in 1 M NaCl, at 10 mV and was permeable to both large cations and anions. In the maximal conducting state, the permeability ratios for Na(+), acetylcholine(+), dopamine,(+) and glutamate(-), relative to Cl(-), were estimated to be 0.73, 0.6, 0.44, and 0.4, respectively. In contrast, in the subconducting state, glutamate(-) was impermeable, while the relative permeability to acetylcholine(+) increased and to dopamine(+) remained unchanged. At the high concentrations (0.1-0.5 M) used in the permeability experiments, glutamate eliminated the bell shape of the voltage dependence of VDAC channel conductance. Glutamate at concentrations of 1 to 20 mM, in the presence of 1 M NaCl, was found to modulate the VDAC channel activity. In single-channel experiments, at low voltages (+/-10 mV), glutamate induced rapid fluctuations of the channel between the fully open state and long-lived low-conducting states or short-lived closed state. Glutamate modification of the channel activity, at low voltages, is dependent on voltage, requiring short-time (20-60 sec) exposure of the channel to high membrane potentials. The effect of glutamate is specific, since it was observed in the presence of 1 M NaCl and it was not obtained with aspartate or GABA. These results suggest that VDAC possesses a specific glutamate-binding site that modulates its activity.