DNA methylation plays an important role in carcinogenesis and aberrant methylation patterns have been found in many tumors. Methylation is regulated by DNA methyltransferases (DNMT), catalyzing DNA methylation. Therefore inhibition of DNMT is an interesting target for anticancer treatment. RX-3117 (fluorocyclopentenylcytosine) is a novel demethylating antimetabolite that is currently being studied in clinical trials in metastatic bladder and pancreatic cancers. The active nucleotide of RX-3117 is incorporated into DNA leading to downregulation of DNMT1, the maintenance DNA methylation enzyme. Since DNMT1 is a major target for the activity of RX-3117, DNMT1 may be a potential predictive biomarker. Therefore, DNMT1 protein and mRNA expression was investigated in 19 cancer cell lines, 26 human xenografts (hematological, lung, pancreatic, colon, bladder cancer) and 10 colorectal cancer patients. The DNMT1 mRNA expression showed large variation between cell lines (100-fold) and the 26 xenografts (1100-fold) investigated. The DNMT1 protein was overexpressed in colon tumours from patients compared to non-malignant mucosa from the same patients (P = 0.02). The DNA methylation in these patients was significantly higher in tumour tissues compared to normal mucosa (P = 0.001). DNMT1 expression in normal white blood cells also showed a large variation. In conclusion, the large variation in DNMT1 expression may serve as a potential biomarker for demethylating therapy such as with RX-3117.