Abstract A transdermal steroidal delivery system usually contains a high concentration of drug to obtain high drug fluxes. The present investigation involved the development of drug-in-adhesive transdermal systems of estradiol using synthesized acrylate copolymer (EA) of 2-ethylhexyl acrylate and acrylic acid. The effect of several variables such as varying drug polymer ratios, effect of Eudragit ® RL PO and Eudragit ® E PO and effect of drying temperatures on prevention of drug crystallization in the formulation matrix was investigated. The systems free from drug crystals were evaluated and compared with a marketed formulation with respect to its skin permeation profile. The optimized formulation was also subjected to accelerated stability testing. Eudragit ® RL PO and Eudragit ® E PO were found to be effective as crystallization inhibitors in the transdermal matrix systems tested. Formulations fabricated with Eudragit ® E PO gave transparent systems with good film properties and a higher skin permeation profile as compared to that of the marketed system. Higher temperature and humidity conditions facilitated the formation of drug crystals, whereas no crystals were observed in the formulation matrix at 23±0.5°C and at 30±1°C for the period of 6 months studied.