Astrocytic glial cells in primary culture from neonatal rat brain possess prominent dipeptidyl peptidase-II activity. This enzyme has been previously isolated and purified from whole brain tissue. The glial enzyme characteristically hydrolyzed glycyl-proline-p-nitroanilide (GPN) substrate to release glycyl-proline dipeptide plus p-nitroaniline products. At the enzyme's optimal pH of 5.4, the activities of other tested amino exopeptidases were virtually zero. At pH greater than 8, activity was less than 2% of the activity at pH 5.4, which suggested a paucity of the related enzyme, dipeptidyl peptidase-IV. No competitive inhibition was observed for glycine, proline nor their permuted dipeptides. Glial dipeptidyl peptidase-II activity was strongly inhibited by Hg2+, while other redox sulfhydryl agents were ineffective. Tested cations did not affect activity, except K+ which was mildly inhibitory. Chelating agents were not inhibitory. Of the peptidase inhibitors tested, only phenylmethylsulfonyl fluoride and puromycin were partially inhibitory. We suggest that dipeptidyl peptidase-II may play a role in glial processing of brain peptides which possess an N-terminal penultimate proline residue.