Phosphodiester linkages, typically generated by action of nucleotidyl transferases, are the backbone of three distinct metabolic scaffolds that function in distinct cellular niches. Both RNA polymerases and DNA polymerases generate internucleotide phosphodiester bonds as the sole covalent links in these informational biopolymers. Essentially every controlled enzymatic manipulation from DNA ligating repair, recombination, and replication, to RNA splicing involve phosphodiester-cleaving (and regenerating) enzymes. The suite of known nucleic acid phosphodiesterases, aka nucleases, number over 100 entities that carry out various regiospecific DNA and RNA phosphodiester bond cleavages, sometimes reversibly. The second major category of phosphodiester metabolites are the cyclic nucleotides that function as second messengers in cell signaling regimes. These include the now classic 3′,5′-cyclic-AMP and cyclic GMP but also the 3′,3′-di-cyclic GMP in bacterial signaling and the 2′,3′-cGAMP signaling molecule in turning on interferon in mammalian antiviral responses to foreign DNA. The action of the various phosphodiesterases that hydrolyze these signaling molecules back to 5′-AMP and 5′-GMP are analyzed. The third category is encompassed by enzymes of the phospholipase C and phospholipase D families that cleave on either side of the phosphodiester linkage in the head group of such membrane lipids as phosphatidylcholines and phosphatidylinositols.