Two novel cDNAs, DNAS1L2 and DNAS1L3, are predicted to encode proteins of 299 and 305 amino acids with 56 and 46% residue identity (71 and 63% similarity), respectively, to deoxyribonuclease I (DNase I). DNAS1L2 is located on a 16p13.3 cosmid, while DNAS1L3 maps to 3p14.3-p21.1 by fluorescence in situ hybridization and by PCR analysis of a radiation hybrid panel. Northern analysis revealed DNAS1L3 expression nearly exclusively in liver, while DNAS1L2 expression was detected in brain by RT-PCR. The previously defined DNL1L or DNAS1L1 is expressed highest in heart and skeletal muscle, while DNase I is expressed in the pancreas, parotid gland, and kidney. Thus, to date, four DNase I-like genes that show different tissue expression patterns are known. A comparison of DNAS1L1, DNAS1L2, and DNAS1L3 with the well-characterized DNase I suggests that the DNAS1L proteins are unlikely to be glycosylated or bind actin; however, catalytic and calcium- and DNA-binding residues are conserved, and potentially cleavable signal peptides are present among all these proteins. This analysis also identifies regions of high conservation among these proteins with no currently assigned function.