Lymph transport is generally accepted to rely upon an intrinsic contractile activity generated locally within and along the walls of lymph vessels. The pumping behaviour of transporting lymphatic vessels resembles that of the gastrointestinal tract where interstitial cells of Cajal (ICC) are critical for coordinating contractile function. Interstitial Cajal-like cells (ICLC) are reminiscent of ICC in their morphology and ultrastructure but are found outside of the gut in other smooth muscle-containing tissues. ICLC have already been demonstrated in several spontaneously active smooth muscle-containing tissues and c-kit positive ICLC have been identified in the subendothelium of sheep mesenteric lymphatics (McCloskey et al. 2002). In many tissues, including animal lymphatic vessels, ICLC are speculated to coordinate electrical activity or act as pacemaker cells. While isolated segments of human thoracic duct (TD) have spontaneous and evoked pump-like contractile activity (Telinius et al. 2010) the anatomical architecture of this tissue is poorly understood. Given the increasing evidence of ICLC populations in many smooth muscle tissues we sought to establish the presence of ICLC in the TD while defining the histology and ultrastructure of this large lymphatic vessel. Thoracic duct tissue was obtained from the mid-thoracic region from >40 adult patients (average age of approximately 60 years; predominantly male) undergoing surgery due to esophageal and cardia cancer at the Department of Cardiothoracic and Vascular Surgery, Aarhus University Hospital, Skejby. The TD is prophylactically ligated and resected from the mid-thoracic region during the surgery; thus the removal of TD tissue does not represent an additional procedure for the patient. The protocol was reviewed and approved by the ethical committee for Region Midtjylland Denmark and the study conducted in accordance with the principles of the World Medical Association Declaration of Helsinki. All patients received information about the study and participated with informed consent. Using conventional histology and immunohistochemistry of thin, fixed preparations and immunofluorescence of whole-mount fixed preparations by confocal microscopy, we investigated the morphology of the TD from the mid-thorax region and the presence and localisation of ICLC protein markers (c-kit, CD34, and vimentin). Transmission electron microscopy was employed to investigate ultrastructure. ICLC were also investigated in live tissue preparations using methylene blue staining as well as with Ca2+-dependent fluorophores and confocal fluorescence microscopy. Methylene blue stained bipolar and stellate shaped cells in the media of the TD. Immunoreactivity for the ICLC protein markers c-kit, CD34 and vimentin in thin sections and whole-mount tissue localised to cells and processes associated with SMC in the media and subendothelium. Loading and imaging of the thoracic duct with Ca2+-dependent fluorophores provided a similar morphological picture as observed with fixed tissues. SMC bundles were observed in multiple orientations; and in association with these bundles, brightly fluorescent small cell bodies and processes were observed. Electron microscopy, the gold standard for identifying ICLC, unequivocally demonstrated the presence of ICLC in the human TD. ICLC with stellate or bipolar appearance were found throughout the TD wall: localised in the subendothelial region as well as in intimate association with SMC throughout the media where they were seen to envelop SMC bundles with their processes. ICLC connected to SMC in the human TD via peg-and-socket junctions and close appositions. While other types of interstitial cells were observed at the EM level, ICLC were predominant and clearly distinguishable at the ultrastructural level by the presence of caveolae, dense bands and a patchy basal lamina. During our initial functional experiments with Ca2+-dependent fluorescence and confocal microscopy, we have observed global Ca2+ events in cells with morphology and localisation suggestive of ICLC in association with spontaneous contractions, potassium-induced depolarisation, and mechanical stretch. Our data demonstrate a novel population of ICLC throughout the human TD from the mid-thorax region. This finding is of particular relevance for future physiological investigations of the pumping activity in human lymphatics as ICLC may represent the initiator or coordinator of this activity.