Elevations of inflammatory cytokines have been implicated in the pathogenesis of experimental autoimmune uveoretinitis (EAU) in rats, although such analysis has relied on indirect methods of assessment such as measurement of mRNA content. In this study, we examined the feasibility of directly measuring cytokine concentrations in intraocular extracts prepared by ultrasonic disruption. Cytokines were measured by ELISA in eyes from EAU-induced Lewis rats immunized with interphotoreceptor retinoid-binding protein (IRBP), and compared to eyes from rats immunized with adjuvant only and from normal rats. Interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-10 (IL-10) were detectable in EAU eyes at near peak inflammation, with IFN-gamma achieving the highest mean concentration (331 pg/ml). In eyes from rats immunized with adjuvant only and in normal eyes, IFN-gamma, TNF-alpha, and IL-10 were nondetectable. IL-2 and IL-4 were detected at significantly lower mean concentrations (32.3 pg/ml and 69.4 pg/ml, respectively) compared to EAU eyes (217 pg/ml and 230 pg/ml, respectively); IL-4 was also detected in eyes from rats immunized with adjuvant alone (141 pg/ml). Thus, a direct method of measuring intraocular cytokine concentrations was successfully applied to reveal an elevation of IFN-gamma, TNF-alpha, IL-2, IL-4, and IL-10 in EAU eyes from rats immunized with IRBP, compared to rats immunized with adjuvant alone and to normal rats. These cytokine elevations reflect the local intraocular environment near peak inflammation, and suggest an important role for these cytokines in the mechanisms of onset and resolution of EAU in rats.