Guanine nucleotide-binding proteins, or G proteins, play an important role in transmitting information from membrane receptors to intracellular effector systems. Activation of G proteins results in the hydrolysis of GTP, and the measurement of GTPase activity represents a means by which the role of G proteins in signal transduction can be investigated. GTPase activity of guinea pig bronchial membranes was measured as the liberation of 32p i from ['y-32p]GTP. GTPase activity was divided into two components, one possessing a high affinity and the other a low affinity for GTP. The contribution of high-and low-affinity GTPase to total hydrolysis was dependent on Mg zΓ·. In the presence of submicromolar Mg 2+, highaffinity GTPase represented 65-80% of all activity, whereas in the presence of t>26 IxM Mg z+, all detectable hydrolysis was due to the low-affinity GTPase. High-affinity GTPase was stimulated by Mg 2Γ· in the 0.15-1. l IxM range (2.5-fold maximal stimulation, apparent Km for Mg 2+ 0.31 txM). Mastoparan (1-100 p~M) caused a concentration-dependent stimulation of highaffinity (but not low-affinity) GTPase (71 ---13% maximal stimulation, ECs0 0.38 IxM), suggesting that high-affinity GTPase may be due to a G protein. Carbachol (10 txM) and fenoterol (10 IxM) had no effect on high-affinity GTP hydrolysis, suggesting that under the conditions described, GTPase activity of bronchial membranes is not activated by muscarinic or 13-adrenergic receptors, respectively.