The manner in which the heterotrimeric G protein complexes G beta(1)gamma(2) and G alpha(i)beta(1)gamma(2) interact with membranes is likely related to their biological function. We combined complementary measurements from sum frequency generation (SFG) vibrational and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy to determine the possible membrane orientations of G beta(1)gamma(2) and the G alpha(i)beta(1)gamma(2) heterotrimer more precisely than could be achieved using SFG alone. The most likely orientations of G beta(1)gamma(2) and the G alpha(i)beta(1)gamma(2) heterotrimer were both determined to fall within a similar narrow range of twist and tilt angles, suggesting that G beta(1)gamma(2) may bind to G alpha(i), without a significant change in orientation. This "basal" orientation seems to depend primarily on the geranylgeranylated C-terminus of G gamma(2) along with basic residues at the N-terminus of G alpha(i), and suggests that activated G protein-coupled receptors (GPCRs) must reorient G protein heterotrimers at lipid bilayers to catalyze nucleotide exchange. The innovative methodologies developed in this paper can be widely applied to study the membrane orientation of other proteins situ.