The passage of endocytosed receptor-bound ligands and membrane proteins through the endocytic pathway of mammalian cells to lysosomes occurs via early and late endosomes. The latter contain luminal vesicles and are also referred to as multivesicular bodies (MVBs). The overall morphology of endosomal compartments is, in major part, a consequence of the many fusion events occurring in the endocytic pathway. Kissing events and direct fusion between late endosomes and lysosomes provide a means of delivery to lysosomes. Using time lapse confocal microscopy we have studied content exchange between lysosomes and endosomes in living cells and found evidence for both these modes of delivery of endosomal content to lysosomal hydrolases [1]. Following fusion in living cells, tubulation and budding events occur that may be part of the process of lysosome re-formation. Fusion of lysosomes with late endosomes results in the formation of a hybrid compartment which acts as a ‘cell stomach’ in which hydrolysis of endocytosed macromolecules occurs and from which lysosomes are re-formed. The formation of hybrid organelles and some aspects of lysosome re-formation have been reconstituted in cell-free systems [2]. Such systems have allowed the identification of some of the biochemical requirements for fusion and lysosome re-formation including the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex required for fusion [3]. The ionic composition of the lumen of endocytic organelles is known to be important for sorting and traffic through the endocytic pathway. Some endocytosed ligands such as low density lipoprotein (LDL) are delivered to lysosomal hydrolases for degradation after dissociation from their receptors in the acidic lumen of the early endosome, a process which also allows subsequent recycling of the empty receptors to the cell surface. Other ligands, such as epidermal growth factor (EGF), remain bound to their receptors which are ubiquitinated and, after endocytosis, are sorted into the luminal vesicles of MVBs. Formation of MVBs and sorting into the luminal vesicles requires a group of cytosolic proteins organised into endosomal sorting complexes required for transport (ESCRT) and probably also a sodium/ proton exchanger which is the mammalian homologue of yeast protein Vps44p. This may be involved in maintaining the ionic balance of the endosome lumen. The fusion of late endosomes with lysosomes and the re-formation of lysosomes from the resultant hybrid organelles both have a requirement for luminal Ca2+ [2]. Thus, fusion was inhibited by BAPTA, but not by EGTA, a chelator with a similar dissociation constant for Ca2+ but an on-rate much slower than BAPTA. Inhibition was reversed by adding additional Ca2+. Fusion was also inhibited by EGTA-AM, a membrane permeable, hydrolysable ester of EGTA. Taken together, the data suggested that it is release of luminal Ca2+ that is required for membrane fusion. . The recovery of electron dense lysosomes from hybrid organelles was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The effect of EGTA-AM suggested that luminal Ca2+ was required for the condensation process necessary for re-formation of electron dense lysosomes. Ca2+ also appears to play an important role in the traffic of some lipids through the late endocytic pathway. Mutations in the gene MCOLN1 result in the lysosomal storage disorder mucolipidosis type IV (MLIV). MCOLN1 encodes mucolipin-1, a lysosomal membrane protein thought to be a cation channel. Aberrant lactosylceramide trafficking through the late endocytic pathway in MLIV cells may be rescued by wild type mucolipin-1 expression but not by lysosome-localised mucolipin-1 mutated in its predicted ion pore-selectivity region [4]. Thus, the Ca2+ content and other ionic composition of endocytic organelles together with the correct localisation of membrane proteins contributing to this composition are essential for sorting and delivery in the endocytic pathway.