Modification of bovine brain G proteins by an N-hydroxysuccinimide ester of biotin has been studied. In the presence of GDP, but in the absence of Mg2+, neither guanine nucleotide binding nor GTPase activity of the protein was altered by modification using less than 1.25 mM biotin derivative with 1 mg/ml G protein. Under these conditions the alpha subunit was modified more extensively than the beta and gamma subunits. However, biotinyl-alpha was less readily bound to streptavidin-agarose than was the less modified beta subunit. Biotinyl-beta gamma was isolated from the modified, intact G protein and further characterized to determine if biotinylation alters its functional properties. Isolated biotinyl-beta gamma and unmodified beta gamma were equivalent based upon: 1) inhibition of the S49 cell membrane adenylyl cyclase, 2) changes in hydrodynamic parameters after being recombined with isolated alpha and treated with guanine nucleotides or complexes of fluoride and aluminum, and 3) competition for isolated alpha binding to biotinyl-beta gamma immobilized previously on streptavidin-agarose. Biotinyl-beta gamma prebound to streptavidin-agarose was 70-100% functional, based upon binding of isolated alpha subunits. Estimates of the affinity of alpha binding to biotinyl-beta gamma indicate that bovine brain alpha 41 has a 10-15-fold higher affinity for beta gamma than does alpha 39. Nonhydrolyzable guanine nucleotides and complexes of fluoride and aluminum decreased binding of either alpha 39 or alpha 41 to biotinyl-beta gamma, and these effects were dependent upon the amount of Mg2+ present. GTP decreased binding of alpha 39, but not alpha 41, to biotinyl-beta gamma. These results indicate that GTP can affect G protein subunit interactions and that its effects do not necessarily require an intact membrane environment or the participation of activating receptors or other membrane-associated proteins. They further indicate that biotinylation of beta gamma does not alter its functional properties and that it can be used for studying G protein subunit interactions.