A rat gene, designated DNaseY, encoding a 36 kDa endonuclease was identified and cloned. Sequence analysis of the cDNA showed it to be the rat homologue of human DNAS1L3. The DNaseY gene product had 42% identity to DNaseI, including conserved critical active site residues, the essential disulfide bridge, the calcium binding domain, and a signal peptide, as well as 2 of the 3 signature boxes. Significantly, DNaseY had 2 nuclear localization signals and was more basic (pI 9.5) than DNaseI (pI 4.8). The DNaseY gene contained a number of exons similar to that of DNaseI, separated by much larger introns, resulting in a gene of >17 kb compared to <4 kb gene of DNaseI. The 36 kDa DNaseY gene product was catalytically inactive but was converted to an active 33 kDa endonuclease following processing of the hydrophobic signal peptide. Antibody generated against peptides representing the predicted amino acid sequence of DNaseY cross-reacted with a 33 kDa nuclear protein which possessed endonucleolytic activity. The enzyme was active over a broad pH range (optimum pH 7-8), was Ca2+/Mg2+-dependent, was inhibited by Zn2+, and was capable of both single- and double-stranded DNA cleavage, producing DNA fragments with 3'-OH ends. Furthermore, the DNaseY gene was expressed constitutively in all cells and tissues tested, but it was not transcriptionally up-regulated in apoptotic cells. All these features were consistent with a role in the early stages of apoptotic DNA fragmentation.