The application of extracellular NAADP to skeletal muscle cells resulted in a dose-dependent increase in cytosolic calcium transients. Within 180 seconds approximately 30% of the cells responded. The V-type ATPase inhibitors, bafilomycin A1 (Baf) and concanamycin A1 (Con), are widely used to inhibit NAADP-triggered calcium signals. However, by preventing lysosomal acidification calcium loading of these organelles is also inhibited. Accordingly, we determined calcium transients triggered by 100 nM Baf or Con. Interestingly, by the co-administration of extracellular NAADP with Baf or Con calcium transients were suppressed to basal level. The kinetics of lysosome destruction by Baf or Con were paralleled by “cell shrinking” and acidification. Beside these short-term effects, after 24 hours exposure caspase 3 activity and pre-G1 DNA fragmentation was already observed with 50 nM Baf. Conversely, autophagy was not induced.