Poly-[N-(2-hydroxyethyl)-l-glutamine] (PHEG) prodrugs of the antitumour antibiotic mitomycin C (MMC) were synthesised using peptidyl spacers, tri- and tetrapeptides, to link the drug to the macromolecular carrier. The relationship between the length and composition of the oligopeptide spacer and the rate of drug release was studied by incubation in buffers, serum and in the presence of enzymes (lysosomal enzymes and collagenase IV). It was observed that tetrapeptide-based conjugates generally release MMC more effectively than tripeptide derivatives. Conjugates having a terminal glycine in the spacer are less stable to hydrolysis than those with a terminal hydrophobic amino acid both in buffer and in serum. The gly-phe-ala-leu conjugate released MMC very rapidly in the presence of both lysosomal enzymes and collagenase IV. Biological experiments indicate that PHEG-MMC conjugates act as prodrugs of MMC: cytotoxicity was observed after hydrolytic release of the active compound in vitro. In vivo studies of P388 solid tumour-bearing mice suggest that conjugates which release MMC slowly may be more effective in inhibiting tumour growth and prolonging animal lifespan. Preliminary in vivo bone marrow toxicity studies indicate that PHEG-MMC prodrugs are less myelosuppressive than free MMC.