Human Ape1 is a multifunctional protein with a major role in initiating repair of apurinic/apyrimidinic (AP) sites in DNA by catalyzing hydrolytic incision of the phosphodiester backbone immediately adjacent to the damage. Besides in double-stranded DNA, Ape1 has been shown to cleave at AP sites in single-stranded regions of a number of biologically relevant DNA conformations and in structured single-stranded DNA. Extension of these studies has revealed a more expansive repertoire of model substrates on which Ape1 exerts AP endonuclease activity. In particular, Ape1 possesses the ability to cleave at AP sites located in (i) the DNA strand of a DNA/RNA hybrid, (ii) "pseudo-triplex" bubble substrates designed to mimic stalled replication or transcription intermediates, and (iii) configurations that emulate R-loop structures that arise during class switch recombination. Moreover, Ape1 was found to cleave AP-site-containing single-stranded RNA, suggesting a novel "cleansing" function that may contribute to the elimination of detrimental cellular AP-RNA molecules. Finally, sequence context immediately surrounding an abasic site in duplex DNA was found to have a less than threefold effect on the incision efficiency of Ape1, and ATP was found to exert complex effects on the endonuclease capacity of Ape1 on double-stranded substrates. The results suggest that in addition to abasic sites in conventional duplex genomic DNA, Ape1 has the ability to incise at AP sites in DNA conformations formed during DNA replication, transcription, and class switch recombination, and that Ape1 can endonucleolytically destroy damaged RNA.