The effects of extracellular ATP and other nucleotides on the cytosolic free Ca2+ concentration ([Ca2+]i) have been studied in single primary human hepatocytes and in human Hep G2 and HuH-7 hepatoma cells. ATP, adenosine 5′- O-(3-thiotriphosphate) (ATPγS), and UTP caused a concentration-dependent biphasic increase in [Ca2+]iwith an initial peak followed by a small sustained plateau in most cells. In some cells, however, repetitive Ca2+ transients were observed. The rank order of potency was ATP ≥ UTP > ATPγS, and complete cross-desensitization of the Ca2+responses occurred between ATP and UTP. The initial transient peak in [Ca2+]iwas resistant to extracellular Ca2+ depletion, which demonstrates mobilization of internal Ca2+ by inositol 1,4,5-trisphosphate whose formation was enhanced by ATP and UTP. In contrast, the sustained plateau phase required influx of external Ca2+. Ca2+ influx occurs most likely through a capacitative Ca2+ entry mechanism, which was shown to exist in these cells by experiments performed with thapsigargin. On the molecular level, specific mRNA coding for the human P2Y1, P2Y2, P2Y4, and P2Y6 receptors could be detected by RT-PCR in Hep G2 and HuH-7 cells. However, ADP and UDP, which are agonists for P2Y1 and P2Y6 receptors, respectively, caused no changes in [Ca2+]i, demonstrating that these receptors are not expressed at a functional level. Likewise, α,β-methylene-ATP, β,γ-methylene-ATP, AMP, and adenosine were inactive in elevating [Ca2+]i, suggesting that the ATP-induced increase in [Ca2+]iwas not caused by activation of P2X or P1 receptors. Thus, on the basis of the pharmacological profile of the nucleotide-induced Ca2+-responses, extracellular ATP and UTP increase [Ca2+]iby activating P2Y2 and possibly P2Y4 receptors coupled to the Ca2+-phosphatidylinositol signaling cascade in human hepatocytes. This suggests that extracellular nucleotides from various sources may contribute to the regulation of human liver cell functions.