Arabidopsis thaliana has 54 subtilisin-related protease (subtilase) genes, but what role these proteases play in the plant is largely unknown. Fifty-four gene-specific primer pairs were designed and applied to RT-PCR studies on aboveground tissues of 7-day etiolated seedlings and seedlings grown in a 16-h photoperiod. Thirty-one subtilases were found to be expressed. Nine were expressed equally under those two conditions; eleven were up-regulated under etiolated conditions, ten were down-regulated; and one was expressed irregularly. To study expression at the protein level, a method for antibody-based proteomic analysis of this multi-gene family was initiated. Two polyclonal rabbit antibodies directed to two separate conserved regions on the Arabidopsis subtilases were produced and tested for binding to subtilases. The first set of binding experiments was done on GST-fusions with seven full-length subtilases as well as with eight partial subtilase clones encompassing the appropriate epitopes. The latter were cloned from high-fidelity PCR amplification products. Five of the seven full-length expressed subtilases showed binding to the antibodies. Seven out of the eight partial-length expressed subtilases were also recognized by antibody. Since there are other subtilases that have sequences identical to or closely matching the epitope regions on the expressed proteins, these results indicate that a small number of antibodies can be used to detect a large number of members of this multi-gene family. In actual protein extracts analyzed by SDS-PAGE, the antibodies recognized several proteins in the size range of subtilases. To resolve the proteins, extracts were pre-fractionated on ion-exchange spin columns, followed by separation of the proteins in the subtilase-containing fraction by 2D PAGE. A combined total of 21 spots were detected by the antibodies in western blot analysis of proteins from the etiolated 7-day seedlings and the seedlings grown in a 16-h photoperiod. Using narrow range IPG strips to resolve the spots better, five spots were excised from replicate silver-stained gels and analyzed by peptide mass fingerprinting with tentative identification of the spots as subtilases.