The human lung small cell adenocarcinoma cell line, A549, demonstrates a concentration-dependent rise in [Ca2+]i in response to extracellular nucleotides. The cells show Ca2+ mobilization on addition of various nucleotides, with an order of agonist potency: UTP > or = ATP > ADP > ADP beta S > AMP; adenosine is ineffective. The EC50 values for UTP and ATP are 12.5 +/- 0.4 microM and 18.9 +/- 0.5 microM, respectively. Together, these results are strongly indicative of the P2U subclass being the major nucleotide receptor expressed in these cells. The Ca2+ response was typically biphasic consisting of an initial spike, representing release of Ca2+ from internal stores, and a subsequent plateau representing Ca2+ influx. The majority of cells showed an agonist-induced Ca2+ increase that was unaffected by pretreatment with the Ca(2+)-ATPase inhibitors 2,5-di(tert-butyl)1,4-benzohydroquinone or thapsigargin. Caffeine did not raise [Ca2+]i above basal levels and applied in conjunction with nucleotide did not attenuate the agonist-mediated response. The Ca2+ influx was sensitive to protein kinase C, and agonist addition in the presence of a protein kinase C inhibitor, D-erythrosphingosine, produced a significantly potentiated Ca2+ influx. Furthermore, agonist-mediated Ca2+ influx was abolished in the presence of a protein kinase C activator, phorbol 12,13-dibutyrate. It is concluded that these cells posses a functional P2U receptor that, upon activation, causes Ca2+ mobilization from TBQ and thapsigargin insensitive stores followed by protein kinase C regulated Ca2+ influx.