Most intracellular Ca²⁺ signals result from opening of Ca²⁺ channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca²⁺ into mitochondria. Ca²⁺ channels in lysosomes contribute to endo-lysosomal trafficking and Ca²⁺ signalling, but the role of lysosomal Ca²⁺ uptake in Ca²⁺ signalling is unexplored. Inhibition of lysosomal Ca²⁺ uptake by dissipating the H⁺ gradient (using bafilomycin A₁), perforating lysosomal membranes (using glycyl-L-phenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca²⁺ signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] formation. Bafilomycin A₁ amplified the Ca²⁺ signals evoked by photolysis of caged Ins(1,4,5)P₃ or by inhibition of ER Ca²⁺ pumps, and it slowed recovery from them. Ca²⁺ signals evoked by store-operated Ca²⁺ entry were unaffected by bafilomycin A₁. Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca²⁺ released by Ins(1,4,5)P₃ receptors.