It is essential to understand cellular responses on photodynamic therapy (PDT) to design delivery systems that maximize cytotoxic effects coupled with minimal induction of side effects or protective mechanisms (or both). Here, we investigated mechanisms of toxicity in human ovarian carcinoma A2780 cells treated with structurally diverse N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (P)–mesochlorin e₆ monoethylenediamine (Mce₆) conjugates that possessed differential subcellular accumulation or covalent attachments of photosensitizers (or both). Apoptosis and necrosis were observed after photoactivation, with increased apoptotic responses observed in cells exposed to conjugates possessing Mce₆ linkage via a lysosomally degradable tetrapeptide spacer (HPMA copolymer–Mce₆ conjugates containing Mce₆ bound via glycylphenylalanylleucylglycine [GFLG] linker [P-GFLG-Mce₆], HPMA copolymer–Mce₆ conjugates containing Mce₆ bound via a GFLG spacer and containing nuclear localization sequence, PKKKRKV₁₃₂K(FITC)C [NLS(fluorescein-5-isothiocyanate [FITC])] bound via a thioether linkage [P-NLS(FITC)-GFLG-Mce₆]). Furthermore, the induction of necrosis was more pronounced in cells exposed to conjugates containing both a nuclear localization sequence (NLS) and Mce₆ bound by a degradable linker (P-NLS(FITC)-GFLG-Mce₆). Caspase-independent mechanisms of cell death were identified in cells treated with nuclear-targeted conjugates possessing Mce₆ attached using a nondegradable tether (HPMA copolymer–Mce₆ conjugates containing Mce₆ bound via a GG spacer and containing NLS(FITC) bound via a thioether linkage [P-NLS(FITC)-GG-Mce₆]), whereas low levels of apoptosis and necrosis were detected in cells exposed to photoactivated nontargeted HPMA copolymer–Mce₆ conjugates containing Mce₆ coupled through a nondegradable spacer (HPMA copolymer–Mce₆ conjugates containing Mce₆ bound via GG linker [P-GG-Mce₆]). Variations in gene expression were observed in cells on PDT. Specifically, HSP70 expression was solely detected in cells treated with P-GFLG-Mce₆, whereas the loss of detection of several genes were observed in cells treated with P-NLS(FITC)-GFLG-Mce₆. Variations in cellular responses on PDT using different HPMA copolymer–Mce₆ conjugates will prove useful in the design of optimal HPMA copolymer PDT delivery systems.