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Objectives: To establish a CT lymphangiography method in mice via direct lymph node puncture., Methods: We injected healthy mice (n = 8) with 50 µl of water-soluble iodine contrast agent (iomeprol; iodine concentration, 350 mg/mL) subcutaneously into the left-rear foot pad (interstitial injection) and 20 µl of the same contrast agent directly into the popliteal lymph node (direct puncture) 2 days later. Additionally, we performed interstitial MR lymphangiography on eight mice as a control group. We calculated the contrast ratio for each lymph node and visually assessed the depiction of lymph nodes and lymphatic vessels on a three-point scale., Results: The contrast ratios of 2-min post-injection images of sacral and lumbar-aortic lymph nodes were 20.7 ± 16.6 (average ± standard deviation) and 17.1 ± 12.0 in the direct puncture group, which were significantly higher than those detected in the CT or MR interstitial lymphangiography groups (average, 1.8-3.6; p = 0.008-0.019). The visual assessment scores for sacral lymph nodes, lumbar-aortic lymph nodes, and cisterna chyli were significantly better in the direct puncture group than in the CT interstitial injection group (p = 0.036, 0.009 and 0.001, respectively). The lymphatic vessels between these structures were significantly better scored in direct puncture group than in the CT or MR interstitial lymphangiography groups at 2 min after injection (all p ≤ 0.05)., Conclusions: In CT lymphangiography in mice, the direct lymph node puncture provides a better delineation of the lymphatic pathways than the CT/MR interstitial injection method., Key Points: • The contrast ratios of 2-min post-injection images in the direct CT lymphangiography group were significantly higher than those of CT/MR interstitial lymphangiography groups. • The visibility of lymphatic vessels in subjective analysis in the direct CT lymphangiography group was significantly better in the direct puncture group than in the CT/MR interstitial lymphangiography groups. • CT lymphangiography with direct lymph node puncture can provide excellent lymphatic delineation with contrast being maximum at 2 min after injection., (© 2023. The Author(s).)