Abstract Inhibition of nucleobase transport activities by tyrosine kinase inhibitors (TKIs) has not been studied to date. Since many targeted TKIs are often combined with either capecitabine or 5-fluorouracil (5-FU) in treatment of various advanced cancers, we examined TKI effects on both equilibrative nucleobase transport (ENBT) and sodium-dependent nucleobase transport (SNBT) activities. Our goal was to study effects of TKIs on human ENBT and SNBT activities to explore potential TKI interactions with nucleobase chemotherapy drugs. Effects of TKIs on ENBT activities were investigated in normal human renal proximal tubule epithelial cells (hRPTECs). TKI effects on SNBT activities were assessed in a pig kidney cell line (LLC-PK1). We showed that TKIs inhibited both ENBT and SNBT activities to different extents. Gefitinib inhibited ENBT activity with an IC50 value of 0.7 μM thus indicating high sensitivity of ENBT to inhibition by gefitinib in hRPTECs. Erlotinib > sorafenib > imatinib > sunitinib inhibited ENBT with IC50 values of 15, 40, 60, 78 μM, respectively whereas dasatinib, lapatinib and vandetanib were not inhibitory at concentrations >100 μM. Similar studies in LLC-PK1 cells, which exhibit SNBT activity, showed that vandetanib was the most potent inhibitor followed by sorafenib > erlotinib > gefitinib > sunitinib > imatinib with IC50 values of 14, 25, 28, 40, 47 and 94 μM, respectively whereas dasatinib and lapatinib were not inhibitory at concentrations >100 μM. These results indicate inhibition of both ENBT and SNBT activities by exposure of cells to TKIs. These effects on nucleobase transport activities, which could be direct or indirect, suggest why regimens combining gefitinib and 5-FU have failed. Further studies should test all TKI classes for such inhibitory activities to assess potential effects on combination regimens using TKIs with nucleobase drugs such as 5-FU in cancer treatment. Citation Format: Vijaya L. Damaraju, Michelle Kuzma, Carol E. Cass, Michael B. Sawyer. Inhibition of sodium-independent and sodium-dependent nucleobase transport activities by tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5452. doi:10.1158/1538-7445.AM2015-5452