A rat cDNA-encoding ribosomal protein S3 was used to clone the S3 homolog in Drosophila melanogaster. The Drosophila gene was in turn used to construct fusions between S3 and glutathione S-transferase that were overexpressed in Escherichia coli, purified on affinity columns, and subsequently used for antibody production and biochemical analysis. Antibody specific for S3 was originally tested to determine the subcellular location of S3 by Western (immunoblot) analysis. As expected, the S3 antigen was found associated with purified preparations of ribosomes, but notably the protein was also observed in the nucleus where it was found to be tightly associated with the nuclear matrix. This result, combined with the fact that S3 contains a nuclear localization signal and that the protein shares some homology to a yeast nuclease gene, suggested that S3 might possibly have a role in DNA metabolism. Tests were initially performed to see if S3 contained DNase activity, where it was subsequently determined that the protein specifically cleaved DNA containing an apurinic/apyrimidinic site via a beta-elimination reaction. The DNase activity was inactivated by antibody to S3, indicating that the apurinic/apyrimidinic lyase activity was associated with the Drosophila S3 protein. Taken together, these results suggest that S3 is among a growing class of multifunctional proteins with roles in transcription/translation and DNA repair.