Lung cancer is the most common type of cancer among women and men. Over the last decades, circulating tumour cells have gained more interests, because they provide insight into the genomic and phenotypic landscape of cancer and allow treatment monitoring. DNA methylation is a frequent hallmark of cancer cells. Inter-alpha trypsin heavy chain inhibitor 5, a serine protease, acts as a tumour suppressor gene and low expression of ITIH5 occurs due to hypermethylation of its promoter region.In this study, 38 lung adenocarcinoma patients and 30 healthy controls were included. From each donor blood was drawn and CTCs were enriched using a size-based microfilter. Additionally, we also analysed DNA samples of the primary tumour. From healthy donors, mononuclear cells and cells from the mircofilter (leukocytes) were used as control samples. We examined the methylation status of the ITIH5 promoter region in enriched CTCs, primary tumour, MNCs and leukocytes with quantitative methylation- specific PCR and pyrosequencing using the instruments Pyromark Q24 and Pyromark Q48.Using a size-based microfilter we identified CTCs as nucleated CK+/CD45- cells in 16/38 patients (42.1%). A cut-off value for positive ITIH5 methylation was calculated from blood drawn from HCO for every method. Using the cut-off value, ITIH5 methylation of enriched CTCs was detected in 3/19 of patients when analysed with Pyromark Q48 and 1/32 when analyses with qMSP. With the Pyromark Q24 system, ITIH5 was detected in 0/20 patients. Pyrosequencing revealed that ITIH5 was methylated in 12/21 primary tumour samples. In this study, ITIH5 methylation could rarely be detected in enriched CTCs of lung adenocarcinoma patients. Other methylation markers that are more frequently methylated might be used as alternative markers.