Assessment of the activity of rheumatoid arthritis (RA) is important for the prediction of future articular destruction. ¹⁸F-FDG PET is known to represent the metabolic activity of inflammatory disease, which correlates with the pannus volume measured by MRI or ultrasonography. To evaluate the correlation between ¹⁸F-FDG accumulation and RA pathology, we assessed ¹⁸F-FDG accumulation in vivo using collagen-induced arthritis (CIA) animal models and ³H-FDG uptake in vitro using various cells involved in arthritis. ¹⁸F-FDG PET images of rats with CIA were acquired on days 10, 14, and 17 after arthritis induction. The specimens were subsequently subjected to macroautoradiography, and the ¹⁸F-FDG accumulation was compared with the histologic findings. ³H-FDG uptake in vitro in inflammatory cells (neutrophils, macrophages, T cells, and fibroblasts) was measured to evaluate the contributions of these cells to ¹⁸F-FDG accumulation. In addition, the influence on ³H-FDG uptake of inflammatory factors, such as cytokines (tumor necrosis factor α [TNFα], interleukin 1 [IL-1], and IL-6), and hypoxia was examined. ¹⁸F-FDG PET depicted swollen joints, and ¹⁸F-FDG accumulation increased with the progression of arthritis. Histologically, a higher level of ¹⁸F-FDG accumulation correlated with the pannus rather than the infiltration of inflammatory cells around the joints. In the in vitro ³H-FDG uptake assay, fibroblasts showed the highest ³H-FDG uptake, followed by neutrophils. Although only a small amount of ³H-FDG was incorporated by resting macrophages, a dramatic increase in ³H-FDG uptake in both fibroblasts and macrophages was observed when these cells were exposed to inflammatory cytokines, such as TNFα and IL-1, and hypoxia. Although neutrophils showed relatively high ³H-FDG uptake without activation, no increase in ³H-FDG uptake was observed in response to inflammatory cytokines. ³H-FDG uptake by T cells was much lower than that by other cells. Thus, fibroblasts and activated macrophages contribute to a high level of ¹⁸F-FDG accumulation in the pannus, and hypoxia as well as cytokine stimulation significantly increases ¹⁸F-FDG uptake by these cells. ¹⁸F-FDG accumulation in RA reflects proliferating pannus and inflammatory activity enhanced by inflammatory cytokines and hypoxia. ¹⁸F-FDG PET should be effective for quantifying the inflammatory activity of RA.