In mammals, cellular 5'-nucleotidase (5'-NT) activity (EC 3.1.3.5) encompasses a number of genetically and structurally distinct enzyme forms, either membrane-bound or soluble, mainly cytosolic, that are characterized by broad specificity towards nucleoside 5'-monophosphate substrates differing in base (purine/pyrimidine) and/or sugar (oxy/deoxy-ribose) moieties. In particular, among the cytosolic 5'-NTs active towards pyrimidine nucleotides are cN-III and cdN, ubiquitously distributed in mammalian tissues and treated as a single entity in the early days. cN-III was first linked to a genetic defect, hereditary pyrimidine nucleotidase deficiency, associated to a nonspherocytic hemolytic anemia disorder of still unclear mechanism but metabolically characterized by abnormally high levels of pyrimidine compounds and ribonucleoproteins in erythrocytes, as evidenced by occurrence of basophilic stippling on blood smearings. Since the first review on pyrimidine-specific nucleotidases (Amici, A.; Magni, G., Arch. Biochem. Biophys., 2002, 397(2), 184-190), excellent overviews on the topic appeared in the literature. In the present contribution, the major findings on these two enzymatic proteins, cN-III and cdN, will be described with particular emphasis on the relationships between their structure and function, as well as on their roles in normal and pathological conditions. The catalytic mechanism of both specific hydrolytic and phosphotransferase activities, possessed by both enzymes, will be discussed also in the light of recent solution of both cN-III and cdN three-dimensional structures. This review also focuses on possible therapeutic approaches involving cellular 5'-NTs in detoxifying common antiviral and antineoplastic drugs.