A doxorubicin N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugate (PK1) designed for intracellular lysosomal cleavage following fluid phase pinocytic uptake is currently in early clinical development. This macromolecular prodrug has been encapsulated in niosomes prepared from Span 60/cholesterol/Solulan C24 (a cholesteryl poly-24-oxyethylene ether) (45:45:10). Lipid/surfactant films hydrated with a 3 mg ml −1 solution of PKI using a modification of the dehydration rehydration vesicle (DRV) method gave an encapsulation efficiency of 49.0 ± 1.54%. Vesicle size was 583 ± 191 nm. Span 60 PK1 niosomes were photographed by optical and transmission electron microscopy and found to have a bright fluorescence on the outside of the vesicles and diminished fluorescence in the vesicle core. This is thought to be due to fluorescence quenching of the higher concentration of PK1 inside the niosomes. Span 60 PK1 niosomes stored freeze dried at -40°C, 4°C and 25°C were completely stable and when stored as a liquid suspension at 4°C and 25°C retain 75% of their encapsulated material even after 28 days. Addition of excipients, e.g. polyethylene glycol 8000 and polyvinylpyrrolidone at the rehydration step increased the PK1 encapsulation efficiency to 62% and 65%, respectively. Incubation of PK1 niosomes with plasma resulted in less than 0.02% doxorubicin release after 72 h. When PK1 niosomes were incubated with a lysosomal enzyme preparation doxorubicin release increased to 7% after 72 h. PK1 niosomes have potential for use in targeted cancer chemotherapy.