To determine whether astrocytes release nonprostanoid vasodilators, cells on microcarrier beads were superfused with various agents in the presence of indomethacin, and the effluent was bioassayed and also analyzed for nitric oxide by a chemiluminescence technique. Bradykinin and A23187 induced release of a factor that relaxed arterial rings, an effect that was blocked by hemoglobin. The effluent contained either nitric oxide or a related compound that could be reduced to nitric oxide. Production of this factor was competitively inhibited by the arginine analogs NG-nitro-L-arginine and NG-methyl-L-arginine and could be restored with L-arginine. Quisqualate and norepinephrine were also effective in causing the release of nitric oxide from astroglial cells. Thus, astrocyte-derived relaxing factor has properties similar to those of an endothelium- and neuron-derived relaxing factor.