PC12 cell differentiation was induced by one week of nerve growth factor (NGF) treatment and adenosine A2A receptor expression and activity were analysed. Undifferentiated PC12 cells expressed very high levels of adenosine A2A receptors (approximately equal to 2 pmol/mg) and exhibited strong cyclic AMP (cAMP) responses when stimulated with the selective adenosine A2A receptor agonist 2-[p-(2-carbonylethyl) phenylethylamino-5'-N-ethylcarboxamidoadenosine]. NGF-induced differentiation was accompanied by a down-regulation of adenosine A2A receptors: receptor binding decreased to 500 fmol/mg, immunoreactive A2A receptor protein was decreased by about half and cAMP production was reduced by 60%. In situ hybridization experiments demonstrated a heterogenous distribution of A2A receptor mRNA and a decreased number of strongly labelled cells after NGF treatment. Stimulation of the cells with the non-selective adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA) inhibited NGF-induced mitogen-activated protein kinase activation. These results thus show that NGF-induced differentiation of PC12 cells is accompanied by a decrease in A2A receptor-mediated cAMP accumulation. This might be a way for PC12 cells to counteract an inhibitory effect of A2A receptor activation on some aspects of neurotrophin signalling.