Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 50 to the damage. In addition to the AP-endonuclease activity, APE1 possesses 30-50 exonuclease activity, which presumably is responsible for cleaning up nonconventional 30 ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 30-end nucleotide removal in the 30-50 exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 50 dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 30 end of the short oligonucleotide. The impact of the 30-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 30-50 exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 30 nucleotide removal by APE1 in the 30-50 exonuclease process is the release of the detached nucleotide from the enzyme’s active site.