Dendrimers have potential for delivering chemotherapeutic drugs to solid tumours via the enhanced permeation and retention (EPR) effect. The impact of conjugation of hydrophobic anticancer drugs to hydrophilic PEGylated dendrimer surfaces, however, has not been fully investigated. The current study has therefore characterized the effect on dendrimer disposition of conjugating alpha-carboxyl protected methotrexate (MTX) to a series of PEGylated 3H-labeled poly-L-lysine dendrimers ranging in size from generation 3 (G3) to 5 (G5) in rats. Dendrimers contained 50 surface PEG and 50 surface MTX. Conjugation of MTX generally increased plasma clearance when compared to conjugation with PEG alone. Conversely, increasing generation reduced clearance, increased metabolic stability and reduced renal elimination of the administered radiolabel. For constructs with molecular weights > 20kDa increasing the molecular weight of conjugated PEG also reduced clearance and enhanced metabolic stability but had only a minimal effect on renal elimination. Tissue distribution studies revealed retention of MTX conjugated smaller (G3-G4) PEG570 dendrimers (or their metabolic products) in the kidneys. In contrast, the larger G5 dendrimer was concentrated more in the liver and spleen. The G5 PEG1100 dendrimer was also shown to accumulate in solid Walker 256 and HT1080 tumours, and comparative disposition data in both rats ( 1 to 2 dose/g in tumour) and mice (11 dose/g in tumour) are presented. The results of this study further illustrates the potential utility of biodegradable PEGylated poly-L-lysine dendrimers as long-circulating vectors for the delivery and tumour-targeting of hydrophobic drugs.