Mitotane is currently employed as adjuvant therapy as well as in the medical treatment of adrenocor-tical carcinoma (ACC), alone or in combination with chemotherapeutic agents. It was previously demonstrated that mitotane potentiates chemotherapeutic drugs cytotox-icity in cancer cells displaying chemoresistance due to P-glycoprotein (P-gp), an efflux pump involved in cancer multidrug resistance. The majority of ACC expresses high levels of P-gp and is highly chemoresistent. The aim of our study was to explore in vitro whether mitotane, at con-centrations lower than those currently reached in vivo, may sensitize ACC cells to the cytotoxic effects of doxorubicin and whether this effect is due to a direct action on P-gp. NCI-H295 and SW13 cell lines as well as 4 adrenocortical neoplasia primary cultures were treated with mitotane and doxorubicin, and cell viability was measured by MTT assay. P-gp activity was measured by calcein and P-gp-Glo assays. P-gp expression was evaluated by Western blot. We found that very low mitotane concentrations sensitize ACC cells to the cytotoxic effects of doxorubicin, depending on P-gp expression. In addition, mitotane directly inhibits P-gp detoxifying function, allowing doxorubicin cytotoxic activity. These data provide the basis for the greater effi-cacy of combination therapy (mitotane plus chemothera-peutic drugs) on ACC patients. Shedding light on mitotane mechanisms of action could result in an improved design of drug therapy for patients with ACC.