Chemosensory neurons in the vomeronasal organ (VNO) detect pheromones related to social and reproductive behavior in most terrestrial vertebrates. Current evidence indicate that the chemoelectrical transduction process is mediated by G protein-coupled second messenger cascades. In the present study, attempts were made to identify the G protein subtypes which are activated upon stimulation with urinary pheromonal components. G protein-specific antibodies were employed to interfere specifically with inositol 1,3,4-trisphosphate formation induced by urinary stimuli and to immunoprecipitate G␣-subunits, activation dependently labeled with [␣-32 P]GTP azidoanilide. The results of both experimental approaches indicate that stimulation of female VNO membrane preparations with male urine samples induces activation of G i as well as G o subtypes. Experiments using different fractions of urine revealed that upon stimulation with lipophilic volatile odorants, only G i proteins were activated, whereas G o activation was elicited by ␣ 2u -globulin, a major urinary protein, which is a member of the lipocalin superfamily. Since each G protein subtype is stereotypically coexpressed with one of the two structurally different candidate pheromone receptors (V1R and V2R), the results provide the first experimental evidence that V1Rs coexpressed with G i may be activated by lipophilic probably volatile odorants, whereas V2Rs coexpressed with G o seem to be specialized to interact with pheromonal components of proteinaceous nature.Terrestrial vertebrates detect chemical signals via sensory neurons located in two anatomically distinct systems: the nasal olfactory epithelium (OE) 1 and the vomeronasal organ (VNO). Whereas the main olfactory system is responsible for the "conventional" sense of smell, the VNO appears to specifically detect pheromones, thereby inducing a distinct social or sexual behavior or endocrine response (1-3). In rodents, the major source of phermones seems to be the urine, however, only few volatile (4) and non-volatile (5) urinary substances producing a definite endocrine or behavioral response have been identified. The best characterized non-volatile urinary components in male mouse urine are the "major urinary proteins" (6). Major urinary proteins have been suggested to be involved in puberty acceleration (5,7,8), an endocrinological effect prevented by lesions of the VNO or the accessory olfactory bulb (1). Major urinary proteins of mice and the rat equivalent ␣ 2u -globulin (9) belong to the superfamily of lipocalins, a structurally homologous but diverse family of extracellular proteins, characterized by their ability to bind small, principally hydrophobic molecules (10). Although recent structural studies have characterized the naturally bound volatile substances (11), major urinary proteins lacking the natural volatiles remain active (8) suggesting that proteins themselves act as pheromones.The molecular mechanisms responsible for the detection of pheromones and the signal transduction processes in the VN...