Background: [¹⁸ F]FDG and [¹¹ C]methionine accumulate in lymph nodes draining S. aureus-infected foci. The lymph nodes were characterized by weight, [¹¹ C]methionine-and [¹⁸ F]FDG-positron emissions tomography (PET)/computed tomography (CT), and immunohistochemical (IHC)-staining. Methods: 20 pigs inoculated with S. aureus into the right femoral artery were PET/CT-scanned with [¹⁸ F]FDG, and nine of the pigs were additionally scanned with [¹¹ C]methionine. Mammary, medial iliac, and popliteal lymph nodes from the left and right hind limbs were weighed. IHC-staining for calculations of area fractions of Ki-67, L1, and IL-8 positive cells was done in mammary and popliteal lymph nodes from the nine pigs. Results: The pigs developed one to six osteomyelitis foci. Some pigs developed contiguous infections of peri-osseous tissue and inoculationsite abscesses. Weights of mammary and medial iliac lymph nodes and their [¹⁸ F]FDG maximum Standardized Uptake Values (SUV_FDGmax) showed a significant increase in the inoculated limb compared to the left limb. Popliteal lymph node weight and their FDG uptake did not differ significantly between hind limbs. Area fractions of Ki-67 and IL-8 in the right mammary lymph nodes and SUV_Metmax in the right popliteal lymph nodes were significantly increased compared with the left side. Conclusion: The PET-tracers [¹⁸ F]FDG and [¹¹ C]methionine, and the IHC-markers Ki-67 and IL-8, but not L1, showed increased values in lymph nodes draining soft tissues infected with S. aureus. The increase in [¹¹ C]methionine may indicate a more acute lymph node response, whereas an increase in [¹⁸ F]FDG may indicate a more chronic response.