Abstract  We have previously demonstrated a principal role for nitric oxide (NO) in the endothelium/shear‐dependent regulation of contractility in rat thoracic duct (TD). In this study we tested the hypothesis that cGMP/PKG (cyclic guanosine monophosphate/cGMP‐dependent protein kinase) is central to the intrinsic and extrinsic flow‐dependent modulation of lymphatic contractility. Lymphatic diameters and indices of pumping in isolated, cannulated and pressurized segments of rat TD were measured. The influences of increased transmural pressure (1 to 5 cm H2O) and imposed flow (1 to 5 cm H2O transaxial pressure gradients) on lymphatic function were studied before and after: 1. inhibition of guanylate cyclase (GC) with and without a NO donor, 2. application of stable cGMP analog and 3. inhibition of the cGMP activation of PKG. Additionally, western blotting and immunofluorescent tissue staining were used to analyse the PKG isoforms expressed in TD. We found that the GC inhibitor ODQ induced changes in TD contractility similar to NO synthase blockade and prevented the relaxation induced by the NO donor SNAP. The cGMP analog – 8pCPTcGMP mimicked the extrinsic flow‐induced relaxation in a dose‐dependent manner, whereas treatment with the cGMP/PKG inhibitor – Rp‐8‐Br‐PET‐cGMPS eliminated the intrinsic flow‐dependent relaxation, and largely inhibited the extrinsic flow‐dependent relaxation. Western blotting demonstrated that both PKG‐Iα and Iβ isoforms are found in TD, with ∼10‐times greater expression of the PKG‐Iα protein in TD compared with aorta and vena cava. The PKG‐Iβ isoform expressed equally in TD and vena cava, both being ∼2 times higher than that in the aorta. Immunofluorescent labelling of PKG‐Iα protein in the wall of rat thoracic duct confirmed it's localization inside the TD muscle cells. These findings demonstrate that cGMP is critical to the flow‐dependent regulation of TD contractility; they also indicate an important involvement of PKG, especially PKG‐Iα in these processes and identifies PKG protein as a potential therapeutic target. This article is protected by copyright. All rights reserved.