Arabidopsis has 34 genes encoding proteins related to rapid alkalinization factor (RALF), a peptide growth factor. One of those genes (AtRALF23) is significantly downregulated by brassinolide (BL) treatment of Arabidopsis seedlings or in mutant seedlings expressing a constitutively active form of BES1, a transcriptional effector of the brassinosteroid signaling pathway. Overexpression of AtRALF23 impairs BL-induced hypocotyl elongation in seedlings, and mature overexpressing plants are shorter and bushier. Overexpression of AtRALF23 produces slower growing seedlings, with roots that have reduced capacity to acidify the rhizosphere. AtRALF23 encodes a 138-aa protein, and when an epitope-tagged form (AtRALF23-myc) was expressed in transgenic plants, the protein was processed to release a C-terminal peptide. The presumed junction between the precursor and the processed peptide contains a recognition site for site-1 protease (AtS1P), a plant subtilisin-like serine protease (subtilase). When AtRALF23-myc was expressed in the background of a site-1 protease mutant (s1p-3), or when the AtS1P recognition site (RRIL) was mutated (RR --> GG) and expressed in a wild-type background, the precursor was not cleaved, and the bushy phenotype was not produced. A fluorogenic peptide representing the presumed subtilase recognition site in AtRALF23 was cleaved in vitro by AtS1P. Thus, BL downregulates AtRALF23 expression, presumably relieving the growth-retarding effect of a peptide growth factor, which is processed from a larger precursor protein by AtS1P.