We previously reported that uridine 5'-triphosphate (UTP), a pyrimidine nucleoside triphosphate produced a concentration- and time-dependent increase in the contraction force in isolated right atrial preparations from patients undergoing cardiac bypass surgery due to angina pectoris. The stimulation of the force of contraction was sustained rather than transient. In the present study, we tried to elucidate the underlying receptor and signal transduction for this effect of UTP. Therefore, we measured the effect of UTP on force of contraction, phosphorylation of p38 and ERK1/2, in human atrial preparations, atrial preparations from genetically modified mice, cardiomyocytes from adult mice and cardiomyocytes from neonatal rats. UTP exerted a positive inotropic effect in isolated electrically driven left atrial preparations from wild-type (WT) mice and P2Y-, P2Y- and P2Y-receptor knockout mice. Therefore, we concluded that these P2Y receptors did not mediate the inotropic effects of UTP in atrial preparations from mice. However, UTP (like ATP) increased the phosphorylation states of p38 and ERK1/2 in neonatal rat cardiomyocytes, adult mouse cardiomyocytes and human atrial tissue in vitro. U0126, a MEK 1/2- signal cascade inhibitor, attenuated this phosphorylation and the positive inotropic effects of UTP in murine and human atrial preparations. We suggest that presently unknown receptors mediate the positive inotropic effect of UTP in murine and human atria. We hypothesize that UTP stimulates inotropy via p38 or ERK1/2 phosphorylation. We speculate that UTP may be a valuable target in the development of new drugs aimed at treating human systolic heart failure.