Abstract Background Arginine is an important amino acid for tumor cell growth and development. Argininosuccinate synthetase 1 (ASS1) is a key enzyme required for biosynthesis of arginine. Preclinically, ASS1-deficient tumor cells are particularly sensitive to arginine depletion, and randomised trials exploring this strategy are in progress in hepatocellular carcinoma and mesothelioma using the drug pegylated arginine deiminase (ADI-PEG20). In this study, we determined the metabolic changes induced by ADI-PEG20 treatment in a panel of bladder cancer and mesothelioma cell lines with promoter methylation-dependent silencing of ASS1. We used two cancer cell lines expressing ASS1 as a control. Methods Malignant mesothelioma, ASS1-negative (H2591, MSTO and JU77) and ASS1- positive (H28) cell lines were used. For bladder cancer, ASS1-negative (T24, 253J, UMUC-3) and ASS1-positive (RT112) cell lines were also used. All cells were treated with 750 ng/ml of ADI-PEG20 for 24 hours which induces up to 90% killing of arginine-dependent cell lines. Then, ultra performance liquid chromatography-mass spectrometry (UPLC-MS) technique was employed for untargeted quantitation of the metabolomic changes induced by ADI-PEG20. Results: All cell lines treated with ADI-PEG20 could be clearly discriminated from their untreated control pair when using PCA multivariate analysis. Arginine depletion was noted in all treated cell lines irrespective of ASS1 expression, however the reduction was at least one-log-fold greater in the ASS1-negative tumor cells. Citrulline, n-a-acetylcitrulline, and glutamine were upregulated specifically in ASS1-negative tumor cell lines. The main impact of ADI-PEG20 treatment was on pyrimidine metabolism in the ASS1-deficient tumor cells with upregulation of thymine and downregulation of thymidine, ureidosuccinic acid, uridine monophosphate and 5-hydroxymethyluracil. Notably, we identified that the reduction of the thymidine nucleotide pool was linked to suppression of thymidylate synthetase and dihydrofolate reductase, and paradoxically, reduced uptake of 3H-FLT. Finally, ADI-PEG20 caused variable effects on cytidine, uridine and ornithine levels in different cancer cell lines. Conclusion This study provides an insight into possible metabolic pathways affected by ADI-PEG20. The impact of ADI-PEG20 on thymidine metabolism, in particular, may be employed as a potential biomarker for optimizing the efficacy of ADI-PEG20 in the treatment of arginine auxotophic cancers. Citation Format: Essam A. Ghazaly, Phuong Luong, Malgorzata Chmielewska-Kassasir, Chantelle Hudson, John S. Bomalaski, L Wozniak, Norbert E. Avril, Simon P. Joel, Peter W. Szlosarek. A comprehensive untargeted UPLC-MS based metabolomic analysis of ASS1-deficient solid tumor cell lines treated with arginine deiminase. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1885. doi:10.1158/1538-7445.AM2013-1885