Objective: We investigated the levels of adrenomedullin (AM) system during the process of preadipocyte differentiation and its role in lipid metabolism and cellular signaling mechanism in differentiated adipocytes. Method: We cultured rat preadipocytes and measured the following during the process of differentiation: two molecular forms of AM in the culture medium using a specific immunoradiometric assay and gene expression of AM and its receptor component using RT-PCR analysis. In differentiated adipocytes, we measured the effects of AM on the intracellular cAMP level, lipolysis, glucose incorporation, and the protein levels. Results: Two molecular forms of AM were secreted into the medium and the AM-mature/AM-total ratio was increased after 6 days of differentiation. Cultured rat preadipocytes highly expressed the genes of AM and its receptor components at day 1 and they increased at day 10. Administration of AM to preadipocyte increased the number of Oil Red O-positive adipocytes and spectrophotometric absorbance of Oil Red O. AM dose-dependently increased cAMP level and lipolysis and its effect was blocked by CGRP(8-37). Isoproterenol increased lipolysis and AM had additive effects on isoproterenol-induced lipolysis. KT5720 and U0126 significantly inhibited the AM-induced lipolysis, whereas KT5720, but not U0126 significantly inhibited the isoproterenol-induced lipolysis. AM increased glucose incorporation and its effect was blocked by wortmannin. Western blot analysis revealed that AM increased phospho PKA, ERK and Akt. Conclusion: These results indicate that AM and its receptor component are highly expressed in cultured adipocytes and may play a role in lipid metabolism via different signaling pathway.