Abstract Tumors deficient in argininosuccinate synthetase-1 (ASS1), a key enzyme involved in arginine synthesis, are auxotrophic for arginine and sensitive to amino acid deprivation. Currently, several trials are testing the arginine-depleting agent, pegylated arginine deiminase (ADI-PEG20) in patients with cancer, including a randomised phase II multicenter UK study in patients with ASS1-deficient mesothelioma (NCT01279967). Here, we sought to identify key pathways involved in the mechanism of action of ADI-PEG20 using a panel of ASS1-deficient malignant mesothelioma (MPM) cell lines as our model. Epithelioid (2591), biphasic (MSTO) and sarcomatoid (JU77) mesothelioma cells were exposed to ADI-PEG20 and analysed using Affymetrix gene expression profiling with validation of candidate genes by qPCR and western blotting, and metabolic studies by LC-MS and 1H-NMR. ADI-PEG20 modulated several metabolic genes involved in nucleotide synthesis in the MPM cell lines. We identified suppression of ribonucleotide reductase (M1 and M2 isoforms), thymidylate synthase and dihydrofolate reductase by 24hrs, the latter enzymes being known targets of pemetrexed, a multitargeted antifolate used in the first-line therapy of mesothelioma. Moreover, metabolic profiling revealed upregulation of several modified nucleosides, including ribothymidine and downregulation of the nucleotide, guanosine monophosphate. Lastly, in vitro studies confirmed potentiation between ADI-PEG20 and pemetrexed and sequential therapy led to reduced tumor growth in vivo. In conclusion, arginine depletion modulates several key enzymes involved in nucleotide synthesis, providing a rationale for combining ADI-PEG20 with pemetrexed-containing regimens in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B139.