Elucidating mechanisms by which Ca2+ signals are generated by monocytes is important for understanding monocyte function in health and disease. We have investigated mechanisms underlying Ca2+ signals generated following disruption of lysosomes by exposure to the cathepsin C substrate glycyl-L-phenylalanine-β-napthylamide (GPN). Exposure to 0.2 mM GPN resulted in robust increases in the intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Ca2+. The response was antagonised by thapsigargin and evoked capacitative Ca2+ entry. Dantrolene-sensitive Ca2+ responses were observed at higher concentrations of GPN (0.4 mM) but not at 0.2 mM. Strikingly, GPN-evoked Ca2+ responses and β-hexosaminidase secretion were inhibited by the ATPase/ADPase apyrase. Simultaneous measurement of [Ca2+]i and extracellular ATP revealed a concomitant secretion of ATP during GPN-evoked Ca2+ signalling. Furthermore, the ability of GPN to raise [Ca2+]i was inhibited by P2Y receptor antagonists or by inhibiting vesicular exocytosis with N-ethylmaleimide (NEM). NEM treatment was associated with an inability of GPN to trigger ATP secretion, a drop in baseline [Ca2+]i and reduction in extracellular ATP concentration. Antagonism of purinergic signalling also caused a reduction in baseline [Ca2+]i. In summary, these data suggest that P2Y receptor activation contributes significantly to GPN-evoked Ca2+ signalling, and that constitutive secretion of lysosomal ATP is a major determinant of Ca2+ homeostasis in monocytes. Lysosomal Ca2+ stores can communicate with ER Ca2+ stores either directly through activation of ryanodine receptors, or indirectly through release of ATP and engagement of P2Y receptors.