The concentrations of gamma-glutamylglutamate, gamma-glutamylglutamine, gamma-glutamylcysteine, glutamate, aspartate, glutamine, cyst(e)ine and glutathione (including disulfides) were determined by HPLC analysis of both the tissue and the surrounding medium of incubated rat hippocampal slices. High potassium concentrations (50 mM; 2 x 4 min) increased the medium concentration of gamma-glutamylglutamate (maximal net efflux 0.07 +/- 0.06 pmol/mg protein/min; n = 8 +/- SD) with a relative time delay compared to the increase in glutamate (maximal net efflux 264 +/- 88 pmol/mg protein/min). Release of gamma-glutamylcysteine, the glutathione precursor, demonstrated an immediate response and gradually approached prestimulus levels (maximal net efflux 0.36 +/- 0.13 pmol/mg protein/min). Addition of acivicin (0.2 mM), a gamma-glutamyl transpeptidase (EC 2.3.2.2.) blocker, during preincubation for 45 min reduced the tissue concentrations (pmol/mg protein) of gamma-glutamylglutamate (19.4 +/- 8.2 (control) vs. 5.8 +/- 3.6 (+ acivicin)), gamma-glutamylglutamine (40.3 +/- 6.7 vs. 25.7 +/- 4.2 pmol/mg protein), glutamine (9.9 +/- 2.0 vs. 4.6 +/- 1.2 nmol/mg protein) and cysteine (1.0 +/- 0.2 vs. 0.56 +/- 0.18 nmol/mg protein). Incubation with acivicin (0.2 mM) reduced the net efflux of gamma-glutamylglutamine (0.79 +/- 0.19 vs. 0.21 +/- 0.07 pmol/mg protein/min) whereas that of the glutathione was increased (4.7 +/- 1.0 vs. 20 +/- 3 pmol/mg protein/min). The medium concentrations of glutamate in both low and high potassium were unaffected by acivicin, while the high potassium induced increase in gamma-glutamylglutamate was blocked. The results demonstrate differential efflux patterns of gamma-glutamyl dipeptides from brain slices and show that in vitro the activity of gamma-glutamyl transpeptidase regulates extracellular concentrations of glutathione, gamma-glutamylglutamine and gamma-glutamylglutamate.