Lipid modifications that may be introduced into several subunits of G proteins were explored by in vitro translation of recombinant mRNAs in reticulocyte lysates. In agreement with studies by others, myristic acid was incorporated into alpha i's and alpha o, but not alpha s, beta, or gamma's. In contrast, mevalonate (Mev) was incorporated only into gamma-subunits. Both, the gamma-subunit of transducin (gamma T) and that of other G proteins (gamma G) were modified by the lysates but with different characteristics. Labeled gamma T was unstable and was rapidly proteolyzed. Labeled gamma G was stable. The Mev-derivative in gamma G was sensitive to methyliodide and, after cleavage and chromatographic analysis, comigrated with the C20 polyisoprenol geranylgeraniol. This indicated that gamma G had been geranylgeranylated and that this polyisoprenoid was attached to the protein through a thioether linkage. It is thought that polyisoprenylation is defined by the COOH-terminal sequence Cys-A-A-X, where A is an aliphatic acid and X is any amino acid. Replacement by mutation of the Cys of the COOH-terminal -Cys-Ala-Ile-Leu sequence of gamma G with Ser abolished Mev incorporation, suggesting this Cys as the site of attachment of the geranylgeranyl moiety. Yet, Mev incorporation was less than 10% as much into gamma G with the Cys-A-A-X sequence -Cys-Ala-Ile-Trp. Consistent with geranylgeranylation, the C15 farnesyl moiety of farnesyl pyrophosphate was not incorporated into gamma G unless the incubations were fortified with Mev. In contrast, the farnesyl moiety was incorporated in an Mev-independent manner into gamma T (COOH terminus: -Cys-Val-Ile-Ser) and c-Ha-ras (COOH terminus: -Cys-Val-Leu-Ser) which are both farnesylated rather than geranylgeranylated. Thus, 1) separate enzymes appear to be involved in transferring farnesyl and geranylgeranyl groups to proteins, 2) structural factors other than the CAAX box contribute to the activity of the polyisoprenylating enzymes, and 3) this type of lipidation may be part of a proteolytic signaling system. Polyisoprenylation, which increases hydrophobicity of the derivatized protein, may play a role in anchoring not only ras but also G proteins to membranes.