Signal transduction via guanine nucleotide binding proteins (G proteins) is involved in cardiovascular, neural, endocrine, and immune cell function. Regulators of G protein signaling (RGS proteins) speed the turn-off of G protein signals and inhibit signal transduction, but the in vivo roles of RGS proteins remain poorly defined. To overcome the redundancy of RGS functions and reveal the total contribution of RGS regulation at the Galpha(i2) subunit, we prepared a genomic knock-in of the RGS-insensitive G184S Gnai2 allele. The Galpha(i2)(G184S) knock-in mice show a dramatic and complex phenotype affecting multiple organ systems (heart, myeloid, skeletal, and central nervous system). Both homozygotes and heterozygotes demonstrate reduced viability and decreased body weight. Other phenotypes include shortened long bones, a markedly enlarged spleen, elevated neutrophil counts, an enlarged heart, and behavioral hyperactivity. Heterozygous Galpha(i2)(+/G184S) mice show some but not all of these abnormalities. Thus, loss of RGS actions at Galpha(i2) produces a dramatic and pleiotropic phenotype which is more evident than the phenotype seen for individual RGS protein knockouts.