Apurinic/apyrimidinic (AP) endonuclease 1 (Ape1) is an essential DNA repair protein that plays a critical role in repair of AP sites via base excision repair. Ape1 has received attention as a druggable oncotherapeutic target, especially for treating intractable cancers such as glioblastoma. The goal of this study was to identify small-molecule inhibitors of Ape1 AP endonuclease. For this purpose, a fluorescence-based high-throughput assay was used to screen a library of 60,000 small-molecule compounds for ability to inhibit Ape1 AP endonuclease activity. Four compounds with IC(50) values less than 10 microM were identified, validated, and characterized. One of the most promising compounds, designated Ape1 repair inhibitor 03 [2,4,9-trimethylbenzo[b][1,8]-naphthyridin-5-amine; AR03), inhibited cleavage of AP sites in vivo in SF767 glioblastoma cells and in vitro in whole cell extracts and inhibited purified human Ape1 in vitro. AR03 has low affinity for double-stranded DNA and weakly inhibits the Escherichia coli endonuclease IV, requiring a 20-fold higher concentration than for inhibition of Ape1. AR03 also potentiates the cytotoxicity of methyl methanesulfonate and temozolomide in SF767 cells. AR03 is chemically distinct from the previously reported small-molecule inhibitors of Ape1. AR03 is a novel small-molecule inhibitor of Ape1, which may have potential as an oncotherapeutic drug for treating glioblastoma and other cancers.