The ATP-dependent proton pump V-ATPase ensures low intralysosomal pH, which is essential for lysosomal hydrolase activity. Based on studies with the V-ATPase inhibitor BafilomycinA1, lysosomal acidification is also thought to be required for fusion with incoming vesicles from the autophagic and endocytic pathways. Here we show that loss of V-ATPase subunits in the Drosophila fat body causes an accumulation of non-functional lysosomes, leading to a block in autophagic flux. However, V-ATPase-deficient lysosomes remain competent to fuse with autophagosomes and endosomes, resulting in a time-dependent formation of giant autolysosomes. In contrast, BafilomycinA1 prevents autophagosome-lysosome fusion in these cells, and this defect is phenocopied by depletion of the Ca(2+) pump SERCA, a secondary target of this drug. Moreover, activation of SERCA promotes fusion in a BafilomycinA1-sensitive manner. Collectively, our results indicate that lysosomal acidification is not a prerequisite for fusion, and that BafilomycinA1 inhibits fusion independent of its effect on lysosomal pH.