1. Spinal prostanoids are implicated in the development of thermal hyperalgesia after peripheral injury, but the specific prostanoid species that are involved are presently unknown. The current study used an in vitro spinal superfusion model to investigate the effect of substance P (SP), N-methyl-d-aspartate (NMDA), and capsaicin on multiple prostanoid release from dorsal spinal cord of naive rats as well as rats that underwent peripheral injury and inflammation (knee joint kaolin/carrageenan). 2. In naive rat spinal cords, PGE2 and 6-keto-PGF1alpha, but not TxB2, levels were increased after inclusion of SP, NMDA, or capsaicin in the perfusion medium. 3. Basal PGE2 levels from spinal cords of animals that underwent 5-72 h of peripheral inflammation were elevated relative to age-matched naive cohorts. The time course of this increase in basal PGE2 levels coincided with peripheral inflammation, as assessed by knee joint circumference. Basal 6-keto-PGF1alpha levels were not elevated after injury. 4. From this inflammation-evoked increase in basal PGE2 levels, SP and capsaicin significantly increased spinal PGE2 release in a dose-dependent fashion. Capsaicin-evoked increases were blocked dose-dependently by inclusion of S(+) ibuprofen in the capsaicin-containing perfusate. 5. These data suggest a role for spinal PGE2 and NK-1 receptor activation in the development of hyperalgesia after injury and demonstrate that this relationship is upregulated in response to peripheral tissue injury and inflammation.