In transfected cells, the P2Y₁₄ receptor reportedly couples to pertussis toxin-sensitive G_i/o-proteins. However, the functional coupling of endogenously expressed P2Y₁₄ receptors to the inhibition of adenylyl cyclase activity has not been reported. Therefore, the primary aim of this study was to investigate the effects of uridine 5′-diphosphoglucose (UDP-glucose) on forskolin-stimulated cyclic AMP (cAMP) accumulation in two cell lines that reportedly express P2Y₁₄ receptor mRNA, namely human neuroblastoma SH-SY5Y cells and human astrocytoma U373 MG cells. In U373 MG cells, UDP-glucose inhibited forskolin-stimulated cAMP accumulation in a concentration-dependent manner (pEC₅₀=4.5±0.3). Furthermore, treatment with pertussis toxin abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation in U373 MG cells. In SH-SY5Y cells, UDP-glucose had no significant effect on forskolin-stimulated cAMP accumulation. To confirm the expression of P2Y₁₄ receptor mRNA in U373 MG and SH-SY5Y cells, we performed reverse transcriptase polymerase chain reaction (RT-PCR) analysis. However, RT-PCR did not detect the expression of P2Y₁₄ receptor mRNA in SH-SY5Y cells or surprisingly in U373 MG cells. In conclusion, we have shown that although UDP-glucose inhibits forskolin-stimulated cAMP accumulation in human U373 MG astrocytoma cells, we did not detect P2Y₁₄ receptor mRNA in these cells. These results would suggest that the effects of UDP-glucose in U373 MG cells are independent of P2Y₁₄ receptor expression. Thus, results obtained with UDP-glucose should be interpreted with caution, since they clearly may not necessarily reflect the involvement of the P2Y₁₄ receptor.