Abstract The effect of intrathecal (i.t.) injection of the cytokine interferon-γ (IFN-γ) on the spinal nociceptive flexor reflex was examined in decerebrate, spinalized, unanesthetized rats. IFN-γ elicited an initial intense, brief facilitation of the flexor reflex followed by a sustained reflex facilitation lasting 40 ± 5 min (range 20–65 min). The initial and prolonged reflex facilitations by IFN-γ were partially and totally blocked, respectively, by i.t. pretreatment with nitro- l-arginine-ester, an inhibitor of nitric oxide synthase, at doses which did not influence spinal cord blood flow. Spinal application of IFN-γ produced powerful and prolonged facilitation of the flexor reflex, possibly reflecting a hyperalgesic action of this cytokine. The facilitatory effect of IFN-γ was mediated, at least in part, by the activation of the l-arginine-nitric oxide pathway. Thus, IFN-γ released in the CNS may participate in eliciting pain and hyperalgesia in infectious or neuroinflammatory diseases where there is increased production of this cytokine.