Drug-protein binding is one of the essential factors in analyzing pharmacokinetics, because it can affect the distribution, metabolism and excretion of a drug within the body. It is also known that one drug can compete with a second drug for the drug binding site on serum proteins. This drug substitution phenomenon can alter the drug concentration level in blood, causing serious side effects in some cases. This effect may be responsible for the recent observations of increased estrogenic effects of estrogenic compounds, either synthetic or environmental. A large number of small molecules, including many drugs, bind reversibly to human serum albumin (HSA, Mr 65K), the most abundant protein in the circulatory system. Some compounds such as penicillinase, acyl glucuronides, and glucose bind covalently to HSA via acyl migration or via the formation of a Schiff's base at lysine or tyrosine residues. There are five drug binding sites known on HSA, Ia, Ib, Ic, II and III, so that competitive binding between various drugs may be an important factor for drugs that can bind to HSA. Because of the potential complexity of drug binding to HSA, it is proposed that this is a good model system for applying mass spectrometric techniques to complexes between proteins and small molecules. The first examples to be explored are the covalent binding of KW-2149 and diazepam. KW-2149 is a mitomycin C derivative that exhibits a broad spectrum antitumor activity in experimental tumor models including mitomycin C-resistant tumors. This drug contains a disulfide linkage. One of the major metabolites of KW-2149 is the HSA conjugate. We have prepared and purified the HSA:KW-2149 conjugate and then subjected the conjugate to tryptic digestion in parallel with unconjugated HSA. The trypsin reaction mixtures were separated by HPLC and the various fractions collected. MALDI/MS analysis of the two reactions showed that there were two peptides in the digest of the conjugate that were not present in unconjugated HSA. These two peptides have been identified as tryptic fragment 5 that is conjugated through a disulfide linkage to either half of the KW-2149 moiety and Cys 34 of HSA.