The trans-prenyltransferase activity present in rat liver microsomes was investigated using an in vitro system. Geranyl-PP, but not farnesyl-PP is utilized as substrate. The pH optimum is at 8.0, and Mn2+ and Mg2+ activate, while Zn2+ completely inhibits the enzyme. Digitonin, taurodeoxycholate, and Tween 80 increase this activity, whereas deoxycholate and SDS are inhibitory. In contrast to the cis-prenyltransferase, the trans-prenyltransferase is not dependent on cytosolic protein factors. The trans-prenyltransferase is present at the cytoplasmic surface of rough and smooth microsomes. Solanesyl-PP and all-trans-geranylgeranyl-PP inhibit the transferase activity but poly-cis-polyprenyl-12-PP, an intermediate in the cis-prenyltransferase reaction, does not. The enzyme reaction gives rise to two products, solanesyl-PP and an unidentified polyprenyl metabolite. Mevinolin treatment distinguishes this enzyme from the cytoplasmic geranylgeranyl-PP synthase. The results demonstrate that rat liver microsomes synthesize solanesyl-PP via a trans-prenyltransferase, which is distinct from cis-prenyltransferase and geranylgeranyl-PP synthase.