Rat liver microsomes catalyzed the formation of A,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate in the presence of Triton X-100. Studies on product specificity using various primers such as Z,E-farnesyl diphosphate, E,E-farnesyl diphosphate, Z,E,E-geranylgeranyl diphosphate, E,E,E-geranylgeranyl diphosphate, Z,E,E,E-geranylfarnesyl diphosphate, and E,E,E,E-geranylfarnesyl diphosphate suggested that the microsomal dehydrodolichyl diphosphate synthase has such properties that it releases Z,E,E-geranylgeranyl diphosphate, the first intermediate, in the reactions with farnesyl diphosphate as the starting primer. Metabolic labeling of rat liver slices with [2-3H]mevalonic acid revealed the accumulation of E,E,E-geranylgeranyl (di)phosphates as well as dolichyl (di)phosphate (C85 and C90) and dehydrodolichol (C85 and C90), but no accumulation of Z,E,E-geranylgeranyl (di)phosphate or E,E-farnesyl (di)phosphate was detected. Microsomal enzyme preparations from mouse liver and hamster liver also produced Z,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate.