We reported previously that cold exposure increases by 2- to 3-times the maximal responsiveness of interscapular brown adipose tissue (IBAT) adenylate cyclase to stimulation by norepinephrine or fluoride and that the sensitizing effect of cold is the result of an increase in neural transmission to the tissue because it is abolished by prior surgical denervation of IBAT. The present report examined further the molecular basis of the sensitization of IBAT adenylate cyclase. Cold exposure increased the maximal responsiveness of adenylate cyclase to stimulation by guanylyl-5'-imidodiphosphate without altering the apparent affinity of the enzyme for the nucleotide. In addition, cold exposure increased the maximal extent of cholera toxin-mediated ADP-ribosylation of the alpha subunit of the stimulatory regulatory protein of adenylate cyclase (Gs alpha) in proportion to the increase in adenylate cyclase activity, but did not alter pertussis toxin-mediated labeling of the inhibitory regulatory protein. Direct measurement of Gs alpha by immunoblotting, however, revealed that sensitized membranes did not contain more Gs alpha protein. These results indicate that neural stimulation of IBAT produced by cold exposure alters the proportion of existing Gs molecules that are functional. The additional observation that sensitized adenylate cyclase activity did not reconstitute into cyc- S-49 lymphoma membranes suggests that the ability of neural stimulation to increase Gs function in IBAT might depend upon the interaction of Gs with factors that are unique to membranes of cold-exposed rats.