The active principles of a monoclonal antibody (791T/36)-human serum albumin-methotrexate (MoAb-HSA-MTX) conjugate have been investigated and identified. This drug-carrier conjugate has previously been shown to be selective for the target cell line, more potent than the 'free' drug, to be internalized into the lysosomes of the cell and to work by a lysosomotropic mechanism. Digestion of MTX-HSA by lysosomal enzymes showed three peaks by HPLC assay. Using authentic standards prepared by solid-phase peptide synthesis, these peaks were identified as 'free' MTX, MTX-Lys (alpha-epsilon) and MTX-Lys (gamma-epsilon). Optimization of the digestion conditions allowed for a maximum total release of MTX-containing material of 5% after 48 h. Of this released material, only 10% was in the form of 'free' MTX. It was shown that increasing the ratio of drug to carrier improved the efficiency of release of drug, and these results were complemented by in vitro cytotoxicity assays. Such a low level of drug release associated with a conjugate which has been shown to be superior, in terms of cytotoxicity, to 'free' drug against the target cell line was an unexpected finding. The consequences of these results are discussed.