Carrier erythrocytes containing drugs, enzymes or peptides can be used as a delivery system that allows changes in the kinetic behaviour and selective biodistribution of the substances encapsulated. Hypotonic dialysis is the method most commonly used in the preparation of carrier erythrocytes, but many factors affect the yield and characteristics of the ghost erythrocytes obtained using this method. This review analyses the factors that affect the performance of carrier erythrocytes prepared by hypotonic dialysis. Factors such as the composition and osmolality range of the hypotonic buffer used, the duration of the hypotonic dialysis, temperature, the volume ratio between the erythrocyte suspension and the dialysis buffer, the inclusion in the process of an annealing phase, the composition and osmolality of the resealing buffer, and the conditions under which the final washing of the erythrocytes is carried out may all affect the morphological properties and the later in vivo behaviour of the ghost erythrocytes obtained. Changes in the yield of the encapsulation process, the in vitro drug or enzyme controlled delivery, the pharmacokinetic properties or the in vivo tissue targeting may be modified depending on the conditions under which the preparation of carrier erythrocytes by hypotonic dialysis is carried out. Chemical alterations to the membrane of carrier erythrocytes obtained by hypotonic dialysis with substances such as glutaraldehyde, band 3 cross-linking reagents, trypsin or NHS-biotin, among others, may affect the release rate of the substances encapsulated and may increase the uptake of cells by macrophages both in vitro and in vivo.