Nucleotides are the building blocks of nucleic acids including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules within virtually all forms of life. While DNA carries the genetic information required for building and maintaining an organism, RNA serves as an intermediary template for protein translation. Nucleic acids derived from pathogens, such as viruses, also represent important molecular patterns that can be sensed by pattern recognition receptors of the innate immune system as danger signals. Engagement of these nucleic acid-sensing receptors initiates activation of signaling cascades in the host immune cells leading to production and secretion of type I interferon (IFN) and other cytokines. The aim of the ensuing antiviral immune response is to eliminate infected cells and to restrict viral spread. As nucleic acid sensors have only limited capacity to differentiate between nonself- and self-DNA or RNA, a type I IFN response can also be initiated by endogenous nucleic acids. Such inappropriate activation of type I IFN can be detrimental to the host by promoting autoinflammation and a loss of immune tolerance leading to autoimmunity. Type I IFN activation induced by immune recognition of self-nucleic acids represents a central pathogenetic mechanism underlying disorders of nucleic acid metabolism and nucleic acid-sensing also referred to as type I interferonopathies. Nucleotides play fundamental roles in cell metabolism. They provide energy in the form of nucleoside triphosphates, function as second messenger in cell signaling, and act as cofactors of enzymatic reactions. Perturbations in adenosine homeostasis leading to downregulation of pyrophosphate (PPi), a key inhibitor of hydroxyapatite growth, underlie disorders of early arterial calcification. The lysosomal equilibrative nucleoside transporter 3 (ENT3) regulates intracellular nucleoside pools and thereby influences the availability of ATP and GTP, synthesis of DNA and RNA, and other metabolic pathways. Its general role in cell homeostasis likely accounts for the pleiotropy of ENT3 deficiency. Activation-induced cytidine deaminase (AID) and uracil-DNA glycosylase (UNG) mediate somatic hypermutation and class-switch recombination at the immunoglobulin gene loci, which represent key events during formation of protective antibodies by B cells. Malfunction of these pathways impedes antibody formation resulting in profound susceptibility to bacterial infections.