PurposeIntravenously (i.v.) administered nanomedicines have the potential for tumour targeting due to the enhanced permeability and retention (EPR) effect, but in vivo tumour models are rarely calibrated with respect to functional vascular permeability and/or mechanisms controlling intratumoural drug release. Here the effect of tumour type and tumour size on EPR-mediated tumour localisation and cathepsin B-mediated drug release was studied.MethodsEvans Blue (10 mg/kg) and an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–doxorubicin (Dox) conjugate (FCE28068) (5 mg/kg Dox-equiv) were used as probes and tumour levels (and Dox release) measured at 1 h after i.v. administration in a panel of murine and human xenograft tumours.ResultsEvans Blue and FCE28068 displayed similar tumour levels in the range of 2–18 % dose/g at 1 h for B16F10 and L1210. Approximately half of the tumour models evaluated exhibited tumour size-dependent accumulation of FCE28068; smaller tumours had the highest accumulation. Administration of free Dox (5 mg/kg) produced tumour levels of <2.5 % dose/g independent of tumour size. Whereas the degree of EPR-mediated targeting showed ~12-fold difference across the tumour models evaluated, Dox release from FCE28068 at 1 h displayed ~200-fold variation.ConclusionsMarked heterogeneity was seen in terms of EPR effect and Dox release rate, underlining the need to carefully calibrate tumour models used to benchmark nanomedicines against known relevant standard agents and for optimal development of strategies for late pre-clinical and clinical development.Electronic supplementary materialThe online version of this article (doi:10.1007/s00280-013-2209-7) contains supplementary material, which is available to authorized users.