The inhibition by bacitracin of the enzymatic dephosphorylation of C(55)-isoprenyl pyrophosphate is abolished by the addition of chelating agents. If, however, the chelating agent is added after a preincubation of bacitracin with a divalent cation and the lipid substrate, then its addition has little effect, indicating that bacitracin, metal ion, and C(55)-isoprenyl pyrophosphate form a complex. Various divalent cations can participate in complex formation, but monovalent cations are ineffective. A direct demonstration of the formation of a complex between the C(55)-isoprenyl pyrophosphate and bacitracin in the presence of metal ions was obtained. Molecular models that show one possible conformation for a complex between bacitracin and the C(55)-isoprenyl pyrophosphate, in which the metal ion acts as a bridge between the two compounds, are presented.