Methotrexate (MTX) is a folic acid antagonist widely used in the treatment of cancer but, like other such agents, has non-specific toxic side effects. With the aim of reducing these toxic effects, MTX was coupled to monoclonal antibodies (MoAb) in one of two ways; either (a) directly using an active ester derivative or (b) via human serum albumin (HSA), to act as an intermediary and so increase the amount of MTX bound. The MTX coupled directly to anti-Ly-2.1 antibody had approximately 10 molecules of MTX per antibody molecule, whereas the HSA coupled material had 24 molecules of MTX per antibody molecule. After coupling MTX directly to antibody there was a loss of potency of the MTX, and MTX-MoAb conjugates were 30-fold less potent than free MTX, although the antibody-coupled material was more specific than free MTX and bound only to the antibody-reactive target cells. By contrast, the MTX-HSA-MoAb conjugates were 3.5 times less potent than free MTX and were 8.5 times more potent than MTX-MoAb conjugates. Thus, by increasing the amount of drug bound to antibody, more toxic conjugates were made--an important principle for the use of such conjugates for the treatment of cancer in man.