Nucleases, in particular ribo- and deoxyribonucleases, are among the least-studied snake venom enzymes. In the present study we have partially purified different nucleases from Vipera lebetina venom. The DNase activity has been proved by DNA degradation both in solution as well as in-gel (zymogram-method). In DNA-containing SDS-PAGE V. lebetina venom exhibits DNA-degrading activity in bands with molecular masses of ˜120, 30–35 and 22–25 kDa. The 120 kDa band corresponds to phosphodiesterase, a 3′, 5′-exonuclease. The endonucleolytic activity of the lower-molecular-mass protein has been confirmed by plasmid degradation and the visualization of the results in agarose gel (with ethidium bromide) electrophoresis. A partial DNA sequence of putative RNase H1 has been determined from the V. lebetina venom gland cDNA library. The translated sequence is similar to the assumed RNase H1 from Crotalus adamanteus (AFJ51163). The RNA/DNA hybrid is hydrolysed by V. lebetina venom and venom fractions. The masses of tryptic peptides from the SDS-PAGE 30–35 kDa band are in concordance with the theoretical peptide masses from the respective translated sequence. For the first time RNase H1-like enzyme activity has been ascertained in snake venom, and sequencing a relevant partial transcript confirmed the identification of this enzyme.