Abstract:The 50‐kilodalton (kDa) assembly polypeptide of bovine brain clathrin coated vesicles (CCVs) is phosphorylated in a cyclic nucleotide‐ and Ca2+‐independent manner and is dephosphorylated by a Mg2+‐ATP‐dependent CCV phosphatase. This report provides evidence for modulation of the phosphorylation reaction of the 50‐kDa assembly polypeptide by phosphorylated clathrin light chain β (pLCβ). In vitro, phosphorylated LCβ inhibits phosphorylation of the 50‐kDa polypeptide in CCVs. Furthermore, incubation of previously phosphorylated 50‐kDa polypeptide in CCVs with phosphorylated LCβ results in a rapid dephosphorylation of the 50‐kDa assembly polypeptide. Both phenomena are time and concentration dependent. Monoclonal antibodies to LCβ prevent the modulatory effect of phosphorylated LCβ on the 50‐kDa assembly polypeptide phosphorylation in CCVs. The results obtained indicate for the first time, to our knowledge, that phosphorylated LCβ has a modulatory role in CCVs. The data also suggest that phosphorylated LCβ promotes activation of a