It has been well documented that Tell positively regulates telomere-end resection by promoting Mre11-Rad50-Xrs2 (MRX) activity, while Rif2 negatively regulates telomere-end resection by inhibiting MRX activity. At uncapped telomeres, whether Tell or Rif2 plays any role remains largely unknown. In this work, we examined the roles of Tel1 and Rif2 at uncapped telomeres in yku70 Delta and/or cdc13-1 mutant cells cultured at non-permissive temperature. We found that deletion of TEL1 exacerbates the temperature sensitivity of both yku70 Delta and cdc13-1 cells. Further epistasis analysis indicated that MRX and Tell function in the same pathway in telomere protection. Consistently, TEL1 deletion increases accumulation of Exo1-dependent telomeric single-stranded DNA (ssDNA) at uncapped telomeres, which stimulates checkpoint-dependent cell cycle arrest. Moreover, TEL1 deletion in yku70 Delta cells facilitates Rad51dependent Y' recombination. In contrast, RIF2 deletion in yku70 Delta cells decreases the accumulation of telomeric ssDNA after 8 h of incubation at the non-permissive temperature of 37 degrees C and suppresses the temperature sensitivity of yku70 Delta cells, likely due to the increase of Mre11 association at telomeres. Collectively, our findings indicate that Tel1 and Rif2 regulate telomere protection at uncapped telomeres via their roles in balancing MRX activity in telomere resection. Copyright (C) 2018, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.