Methotrexate (MTX) has been conjugated to various structurally related, synthetic, branched polypeptides containing a poly(L-Lys) backbone by the aid of water-soluble carbodiimide. The average degree of MTX incorporated was found to be dependent on the size of the polymer and on the identity of the terminal amino acid residue of the side chains. Consequently the average molar substitution ratio was in the range of 4.9-72.0 MTX per carrier molecule. CD spectra of conjugates showed significant differences in solution conformation correlating with the identity of the side-chain-terminating amino acid. Polycationic conjugates XAK-MTX (X = Leu or D-Leu) assumed essentially ordered (helical) secondary structure, while the CD spectrum of the amphoteric conjugate (X = Glu) corresponded to only a partially ordered conformation in PBS. The covalent attachment of MTX to branched polypeptides results in a reduction of drug in vitro cytotoxicity influenced by the carrier structure. Conjugation to amphoteric polymers, depending on the configuration and position of glutamic acid (XAK-MTX vs AXK-MTX type conjugates) resulted in a decrease of anti-791T cell activity. However polycationic conjugates bearing L-Leu at the side chain terminal position (LAK-MTX) produced a compound with cytotoxicity only about 60 times less effective than free MTX. The biodistribution in mice has been characterized by blood clearance, whole-body retention, and tissue distribution 24 h after iv administration. Blood clearance of MTX-branched polypeptides could be significantly prolonged by incorporation of glutamic acid into the side chain.(ABSTRACT TRUNCATED AT 250 WORDS)