The new synthetic tripeptide, p-fluoro-L-phenylalanyl-m-bis-(2-chloroethyl)-amino-L-phenylalanyl-methi onine ethylester hydrochloride, PTT.119, an alkylating agent, is currently undergoing preclinical trials as an antineoplastic agent. The molecular composition, C29H39N4O4SCl2F, was confirmed by desorption chemical ionization mass spectrometry with accurate mass measurement. A high-performance liquid chromatographic technique was developed for the quantification of PTT.119 in cell culture medium and serum. Incubation of 5 X 10(5) mammary tumor cells (MJY-alpha)/ml tissue culture medium with 25 micrograms PTT.119/ml for 60 min (37 degrees C) removed 68% of the tripeptide from the medium. This corresponds to an uptake of 51 fmol PTT.119/tumor cell. Cell death, assessed 5 days after treatment, was directly proportional to the time-dependent removal of PTT.119 from the cell culture medium.