The binding of methotrexate (MTX) and citrovorum factor (CF) to human serum albumin (HSA) was investigated. The affinity constant for MTX was 820 M-1, with 2 binding sites, and for CF 2340 M-1, with 1.5 binding sites. MTX and CF, which are used together in high dose therapy, compete for HSA binding. Competition for HSA binding between MTX and adriamycin, bleomycin and cyclophosphamide, drugs often used in association with MTX in cancer chemotherapy, was also demonstrated. The clinical importance of such competition depends on the drug/protein concentration ratio which is extremely variable.