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The subcellular distribution of Gs alpha was characterized in S49 lymphoma cells with two polyclonal antisera directed against specific COOH- and NH2-terminal epitopes. Nonspecific binding was determined in each subcellular compartment by incubating cyc- S49 cells, known to be deficient in Gs alpha and its mRNA, with primary and secondary antisera. Small proportions of total specific binding sites were localized to the plasmalemma as well as the nuclear envelope. Because of their small size, these compartments contained a high concentration of Gs alpha. However, most of the specific binding sites were found in nonstructured cytoplasm and within the nucleus. Specific binding was abolished or significantly reduced by preincubating primary antisera with their peptide immunogens but not with an irrelevant peptide. Intracellular Gs alpha immunoreactive binding sites did not colocalize with gold-conjugated transferrin in cells preincubated with this ligand to mark a classical endocytotic pathway. The intracellular and intranuclear location of Gs alpha was confirmed with confocal microscopy of S49 cells immunostained with specific primary and fluorescently labeled secondary antibodies. Gs alpha was also detected with immunoblots of proteins extracted from purified S49 cell nuclei. Thus, Gs alpha is abundantly distributed in intracellular and intranuclear sites in S49 cells and occurs in loci distinct from organelles of the transferrin pathway. The substantial intracellular distribution of Gs alpha suggests that Gs may subserve intracellular and, perhaps, intranuclear functions that may be important in proliferating cells.