The effect of beta(3)-adrenoceptor (beta(3)-AR) agonists on adipocytes treated or not treated with signaling modulators has not been sufficiently elucidated. Using rat epididymal adipocytes (adipocytes) labeled with [(32)P]orthophosphate, we found that treatment with the selective beta(3)-AR agonist CL316243 (CL; 1 microM) induces phosphatidylinositol (PI) 3,4,5-triphosphate (PI[3,4,5]P(3)) production and that this response is inhibited by adenosine deaminase (ADA, an adenosine-degrading enzyme; 2 U/ml), pertussis toxin (PTX, an inactivator of inhibitory guanine-nucleotide-binding protein; 1 microg/ml), or wortmannin (WT, a PI-kinase inhibitor; 3 microM). The results showed that CL induced PI(3,4,5)P(3) production in intact adipocytes and that this production was affected by signaling modulators. Taken together, our findings indicate that CL produces PI(3,4,5)P(3) in an ADA-sensitive, PTX-sensitive, or WT-sensitive manner and will advance understanding of the effect of beta(3)-AR agonists on adipocytes.