Saccharomyces cerevisiae redoxyendonuclease (Scr), a homolog of Escherichia coli endonuclease III, was purified from yeast deficient in the major apurinic/apyrimidinic endonuclease, Apnl. Studies of this highly purified preparation of Scr have revealed a number of similarities between this protein and endonuclease III as well as provided further evidence for a common mechanism of action for this class of DNA glycosylase/AP lyases. We have employed a sensitive and specific assay for Scr which utilizes oligonucleotide substrates containing a single 5,6-dihydrouracil base lesion or an abasic site. These substrates were utilized to investigate the mode of action of Scr on damaged DNA and to compare the kinetic properties of the yeast enzyme with its E. coli counterpart. Furthermore, we have identified two distinct genes, SCR1 and SCR2, which encode highly homologous proteins with similar activities in yeast. Analysis of the deduced amino acid sequences of SCR1 and SCR2 suggests that S. cerevisiae possesses two similar enzymes encoded on separate chromosomes: one which bears an Fe-S binding motif, while the other does not. The potential biological roles of these two forms of Scr are discussed.