The purpose of this study was to determine levels of IL-2, -4, -5, -10, TNF-alpha, and IFN-gamma in aqueous humor (AH) from patients with active panuveitis, anterior uveitis (AU), and noninflammatory controls by using a flow cytometric mutiplex array (CBA) and to compare with results from ELISA. Pooled normal AH was spiked with six cytokines at decreasing concentrations for evaluating the CBA. AH was also obtained from 10 controls (cataract patients) and 36 patients with active uveitis. Cell-free supernatants were added to a cocktail of capture beads and detector antibodies or to antibody-coated wells for CBA and ELISA determination, respectively. CBA demonstrated greater sensitivity for detecting IL-4, IL-10, and TNF-alpha than with ELISA. Increased IFN-gamma was detected in both AU and panuveitis groups compared with controls (P < 0.01). IL-10 was higher in the panuveitis group on steroids (P < 0.01). IL-5 was detected in the control (P < 0.01) and AU groups (P < 0.05) but was undetectable in the panuveitis group (n = 10). Correlations between IFN-gamma and IL-10 were found in all uveitis groups (P < 0.01) but not in controls, whereas TNF-alpha correlations with IL-4/IFN-gamma were obtained in controls but not in the uveitis groups (P < 0.01). It was possible to measure cytokines titrated into normal AH specimens by CBA, and a greater number of cytokines were detected with increased sensitivity than with ELISA. Elevated IFN-gamma in active uveitis and decreased IL-5 in posterior uveitis suggest Th1 polarity is more marked, with greater uveal tract involvement. The increased IL-10 in the steroid treated group suggests glucocorticoid-induced IL-10 upregulation.