In the present study, effects of alterations in the chemical structure of polyester-co-polyether (PEPE) dendrimers on the encapsulation and release of methotrexate (MTX) was investigated. A series of PEPE dendrimers of different architecture were synthesized. Biocompatibility of the resulting dendrimers was evaluated in vitro by assessing their cytotoxicity on RAW 264.7 cells using lactate dehydrogenase (LDH) assay. Dendrimers caused no cell death even at the concentration of 250 microg/mL, suggesting that they are acceptable for pharmaceutical applications. They also showed good capacity to encapsulate MTX, with loading as high as 24.5% w/w. Increase in the number of branches and the size of internal voids were shown to enhance the encapsulation. On the other hand, absence of aromatic rings as branching units drastically reduced the loading capacity. Physical entrapment, weak hydrogen bonding and hydrophobic interactions were established to be the mechanisms of encapsulation. Release of MTX was biphasic, which included a burst release in 6h followed by a slower release over a period of 50 or 168 h. Increase in the number of branches profoundly decreased this initial burst release; in contrast, absence of aromatic rings in the dendritic structure resulted in a very rapid release.