Although many physiological changes after space flight have been reported, it is not clear how micrograv- ity influences our bodies. The focus of the present study was to clarify the changes in G-protein-coupled receptor-mediated intracellular signaling, especially Gs-adenylyl cyclase (AC)-adenosine 3� ,5 � -cyclic monophos- phate (cyclic AMP) pathway, under simulated microgravity. Human astrocytoma 1321N1 cells were cultivated under vector-averaged microgravity conditions generated by clinostat rotation (20 rpm) for 24 hr. Isoproterenol, a β-adrenergic agonist and forskolin, a direct AC stimulant, increased intracellular cyclic AMP level in concentration dependent manners, however, both of which response were decreased in cells cultivated in clinostat rotation. While the level of Gαs or intracellular ATP, a substrate for AC, was not changed, the AC activity was significantly low in the membranes of clinostat-rotated cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that AC type 3 (AC3), AC6, and AC9 and to a lesser extent AC7 and AC8 were expressed in 1321N1 cells. Among them, the expression of AC6 mRNA was significantly decreased by clinosta tr otation. These results indicate that intracellular cyclic AMP production by agonists may be decreased via a reduction in AC6 expression under simulated microgravity conditions.