Autophagic cell death has recently received a great deal of attention. However, a dependence of this type of cell death on the actual process of autophagy has only rarely been proven. Indeed, it is important to differentiate between cell death with an accumulation of autophagosomes and cell death actually caused by excessive or inhibited autophagy. The aim of this study was to elucidate the mechanism of action involved in the cytotoxicity of 1-O-hexadecyl-2-O-methyl-3-O-(2'-acetamido-2'-deoxy-beta-D-glucopyranosyl)-sn-glycerol (Gln) and specifically the involvement of autophagy in the effects observed. Our results show that Gln induces cell death associated with large increases in autophagolysosome number and size. However the cell death is independent of autophagy and caspase activation. Instead, Gln leads to lysosomal membrane permeabilisation with a resulting leakage of hydrolases into the cytosol, which are then directly involved in cell death. The increased number of autophagolysosomes, however, is just a side effect of the neutralisation of the lysosomal pH by Gln.