l-Arginine (l-Arg) is an endogenous substrate for nitric oxide synthase (NOS). In the present study, we examined whetherl -tyrosyl-l-Arg (kyotorphin), an endogenous analgesic neuropeptide, might be a substrate for inducible NOS (iNOS) in the brain. Both kyotorphin and l-Arg caused an accumulation of nitrites in lipopolysaccharide (LPS)-treated glial cells cultured from infant rat brains. However, such accumulation of nitrites was not induced by N G -nitro-l-Arg (a NOS inhibitor),l -tyrosyl-d-Arg (d-kyotorphin) or d-Arg.l -Leucyl-l-Arg (an antagonist for kyotorphin receptors) or bestatin (an inhibitor for kyotorphin-hydrolyzing peptidase) did not inhibit the kyotorphin-induced accumulation of nitrites in LPS-treated cells. On the contrary, l-Leucyl-l-Arg caused an accumulation of nitrites in a concentration-dependent manner. The results indicate that nitric oxide (NO) is produced in LPS-treated glial cells directly from kyotorphin through the catalytic action of iNOS.