Leishmania parasites like other kinetoplastids are unable to synthesize purines de novo and so are reliant on a salvage pathway for recycling ribonucleotides. A stage specific class one nuclease enzyme, 3'-Nucleotidase/nuclease, has been implicated in salvage of preformed purines in Leishmania insect stage promastigote via hydrolysis of 3'-nucleotides and nucleic acids. Although a similar activity is known to exist in amastigotes which reside in infected mammalian cells, the homologous gene and the corresponding protein responsible for carrying out this function have not been well characterized. Using primers specific for conserved regions of trypanosomatid class one nucleases, a gene encoding a novel class one nuclease from amastigotes of Leishmania major (LmaC1N) was cloned and sequenced. The coding sequence consists of 951 bp encoding a 316 amino acid protein with a predicted molecular mass of 35,300 Da. Analysis of the deduced amino acid sequence showed that LmaC1N is highly homologous to other class I nucleases and contains all five conserved regions reported for promastigotes 3'-Nucleotidase/nuclease. Analysis by reverse transcriptase polymerase chain reaction and Western blotting demonstrated that expression of LmaC1N gene is regulated in a stage-specific manner. Whereas the gene appeared to be silenced in promastigotes, high level expression in amastigotes implied an important function in support of parasite survival and multiplication in the mammalian cells.