The mechanism of cell death in A2780 human ovarian carcinoma cells induced by free doxorubicin (DOX) and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound DOX [P-(GFLG)-DOX] was investigated. In particular, the involvement of the Fas receptor system in drug-induced apoptosis was evaluated. P-(GFLG)-DOX was shown to effect apoptosis-induced tumor cell death as manifested by positive Annexin V-FITC staining, cleavage of procaspase 3 and its physiological substrate, poly(ADP-ribose) polymerase (PARP), and cleavage of procaspase 8. Using the fluorochrome-labeled caspase inhibitor assay, it was found that both free DOX and P-(GFLG)-DOX activated caspases 3 and 9, but both forms of DOX did not have an effect on the activity of caspase 8, when compared to untreated cells. It was shown that free DOX and P-(GFLG)-DOX upregulated Fas receptor expression at the cell membrane in a time-dependent manner. Triggering the drug-induced Fas receptor with an exogeneous soluble Fas ligand (sFasL) resulted in an increase in the extent of apoptotic cell death, indicating that the Fas signaling pathway remained functionally active. Also, antagonistic anti-Fas ZB4 antibody blocked the increase in the level of apoptosis following the application of sFasL, but did not interfere with drug-induced apoptosis. The study of the functional activity of the Fas receptor and of the activation of the most proximal effector of the caspase cascade, caspase 8, indicated that the Fas receptor pathway was not decisive in the induction of cell death by free DOX and P-(GFLG)-DOX in A2780 cells. This study suggests further investigation of the involvement of the mitochondrial pathway in A2780 cell apoptotic death, induced by free and HPMA copolymer-bound DOX.