Proteins secreted by cancer cells may be useful as tumor markers. We analyzed constituents produced by cells grown in serum-free conditioned medium to avoid confounding factors present in serum. Mass spectrometric techniques were used to obtain expression patterns of constituents between 2000 and 15000 Da from as little as 1 microl of crude sample. This protocol can produce distinctive mass profiles from 16 cell lines within 1 h. Thus, differential display by mass spectrometry will expedite the discovery of peptides specifically secreted by particular cells. These data illustrate the advantage of this strategy over the conventional approach of electrophoretic separation of serum samples.