The 3'-nucleotidase/nuclease (3'-NT/NU) is a surface enzyme unique to trypanosomatid parasites. These organisms lack the pathway for de novo purine biosynthesis and thus are entirely dependent upon their hosts to supply this nutrient for their survival, growth, and multiplication. The 3'-NT/NU is involved in the salvage of preformed purines via the hydrolysis of either 3'-nucleotides or nucleic acids. In Crithidia luciliae, this enzyme is highly inducible. For example, in these organisms purine starvation triggers an approximately 1000-fold up-expression of 3'-NT/NU activity. In the present study, we cloned and characterized a gene encoding this intriguing enzyme from C. luciliae (Cl). Sequence analysis showed that the Cl 3'-NT/NU deduced protein possessed five regions, which we defined here as being characteristic of members of the class I nuclease family. Further, we demonstrated that the Cl 3'-NT/NU-expressed protein possessed both 3'-nucleotidase and nuclease activities. Moreover, we showed that the dramatic up-expression of 3'-NT/NU activity in response to purine starvation of C. luciliae was concomitant with the approximately 100-fold elevation in steady-state mRNA specific for this gene. Finally, results of our nuclear run-on analyses demonstrated that such up-regulation in 3'-NT/NU enzyme activity was mediated at the posttranscriptional level.