BACKGROUND : 2-Methoxyestradiol (2ME2) is an endogenous metabolite of 17-β-estradiol with anti-proliferative and anti-angiogenic properties. Due to 2ME2’s rapid metabolism and low oral bioavailability in in vivo settings, 2ME2 analogues have been designed to alleviate these issues. One of these compounds is 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). A previous work alluded to the ability of ESE-16 to induce autophagic cell death. Therefore, we investigated the mode of action of ESE-16 by studying its effects on autophagy, vesicle formation, and lysosomal organisation. SUMMARY : Vesicle formation and autophagy induction were analysed by transmission electron microscopy (TEM), monodansylcadaverine (MDC) staining and Lysotracker staining, while autophagosome turnover was analysed using microtubule-associated protein 1A/1B-light chain 3 (LC3 lipidation) analysis. MDC staining of acidic vesicles revealed an increase both in the number and size of vesicles after ESE-16 exposure. This was confirmed by TEM. Lysotracker staining indicated an increase in the size of lysosomes, as well as changes in their distribution within the cell. However, autophagy was not induced, since LC3 lipidation did not increase after exposure to ESE-16. KEY MESSAGES : This study showed that ESE-16 exposure leads to the aggregation of acidic vesicles, identified as lysosomes, not accompanied by an induction of autophagy. Therefore, ESE-16 disrupts normal endocytic vesicle maturation likely through the inhibition of the microtubule function.