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Objective To study the feasibility and the potential benefits of defining the internal gross tumor volume (IGTV) of hepatocellular carcinoma (HCC) using contrast-enhanced 4D CT images obtained by combining arterial-phase (AP) contrast-enhanced (CE) 3D CT and non-contrast-enhanced (NCE) 4D CT images using deformable registration (DR). Methods Ten HCC patients who had received radiotherapy beforehand were selected for this study. The following CT simulation images were acquired sequentially: NCE 4D CT in free breathing, NCE 3D CT and APCE 3D CT in end-expiration breath holding. All 4D CT images were sorted into ten phases according to breath cycle (CT00 ~ CT90). Gross tumor volumes (GTVs) were contoured on all CT images and the IGTV-1 was obtained by merging the GTVs in each phase of 4D CT images. The GTV on the APCE 3D CT image was deformably registered to each 4D CT phase image according to liver shape using RayStationTM 3.99.0.7 version treatment planning system. The IGTV-DR was obtained by merging the GTVs after DR on the 4D CT images. Volume differences among the GTVs and between the IGTV-1 and the IGTV-DR were compared. Results The edge of most lesions could be definitively identified using APCE 3D CT images compared to NCE 4D and 3D CT images. The GTV volume on APCE 3D CT images increased by an average of 34.79% (P < 0.05). There was no significant difference among the GTV volumes obtained using NCE 4D and 3D CT images (P > 0.05). The GTV volumes after DR on 4D CT different phase images increased by an average of 36.29% (P < 0.05) as was observed using the APCE 3D CT image (P > 0.05). Lastly, the volume of IGTV-DR increased by an average of 19.91% compared to that of IGTV-1 (P < 0.05). Conclusion NCE 4D CT imaging alone has the potential risk of missing a partial volume of the HCC. The combination of APCE 3D CT and NCE 4D CT images using the DR technique improved the accuracy of the definition of the IGTV in HCC. [ABSTRACT FROM AUTHOR]