Antisera raised against individual sites of histone post-translational modification (PTM) have provided critical insights into the biology of many of these PTMs. However, limitations inherent to immunochemical approaches can skew results obtained with these reagents, possibly leading investigators to misjudge the role of a specific histone PTM in a given process. We have used mass spectrometry in conjunction with cell synchronization, metabolic labeling, RNA interference, and other approaches to show that the SET domain proteins PR-Set7 and Suv4-20 mediate progressive global mono-, di-, and trimethylation of lysine 20 (K20) in newly synthesized histone H4, beginning approximately at the G2/M transition, well after new H4 is deposited in replicating chromatin during S phase. Immunochemical and other approaches have implicated H4-K20 methylation in multiple processes, including gene activation, gene repression, chromatin condensation, S phase progression, mitosis, and DNA-damage checkpoint signaling. Here, we review recent data on the regulation and significance of K20 methylation.