Heterotrimeric G proteins transduce signals from activated transmembrane G protein-coupled receptors to appropriate downstream effectors within cells. Signaling specificity is achieved in part by the specific alpha, beta, and gamma subunits that compose a given heterotrimer. Additional structural and functional diversity in these subunits is generated at the level of posttranslational modification, offering alternate regulatory mechanisms for G protein signaling. Presented here is the identification of a variant of the gamma(2) subunit of G protein heterotrimer purified from bovine brain and the demonstration that this RDTASIA gamma(2) variant, containing unique amino acid sequence at its N terminus, is a substrate for ubiquitylation and degradation via the N-end rule pathway. Although N-end-dependent degradation has been shown to have important functions in peptide import, chromosome segregation, angiogenesis, and cardiovascular development, the identification of cellular substrates in mammalian systems has remained elusive. The isolation of RDTASIA gamma(2) from a native tissue represents identification of a mammalian N-end rule substrate from a physiological source, and elucidates a mechanism for the targeting of G protein gamma subunits for ubiquitylation and degradation.