Multiple lines of evidence implicate the excitatory amino acids (EAAs) (L-aspartate (L-Asp) and L-glutamate (L-Glu) as excitatory transmitters in the spinal cord. The specific objective of this study was to determine whether the EAAs are released from primary afferents. Dorsal root ganglia (DRG) from 2 to 18-day-old rats dissected and cultured for 1-2 weeks were washed in modified Ringers recording solution for a period of 1 h to allow equilibration. The mean +/- S.E.M. baseline concentrations of EAAs recovered during a 5 min interval were 533.29 +/- 65.59 nmol for L-Glu and 106.67 +/- 14.05 nmol for L-Asp. Stimulation of DRG organotypic cultures with potassium resulted in a significant concentration-dependent increase in the release of both EAAs. The concentration of Asp increased to 166 +/- 17% and 203 +/- 13% in response to 5 min exposure of the culture to 25 and 50 mM potassium, respectively. The concentration of Glu increased to 155 +/- 12% and 226 +/- 18% of control in response to the same stimuli. In response to application of 50 mM potassium for 25 min, peak concentrations increased to 465 +/- 53% for Asp and 312 +/- 51% for Glu of the basal concentration. Exposure of the cultures to 1 or 10 microM capsaicin also caused release of both EAAs. The concentrations of Asp and Glu significantly increased to 204 +/- 11% and 165 +/- 15% of basal concentrations, respectively, in response to a 5 min exposure to 1 microM capsaicin. High [K+]e failed to increase the release of EAAs from cultures where DRG cell bodies were removed 72 h prior to release experiments. These results confirm results demonstrating release of EAA from mammalian spinal cord tissues and directly demonstrate for the first time that primary afferent fibers are specifically involved in this release.