The subtilisin-like serine protease SbtMl is strongly and specifically induced during arbuscular mycorrhiza (AM) symbiosis in Lotus japonicus. Another subtilase gene, SbtS, is induced during early stages of nodulation and AM. Transcript profiling in plant symbiosis mutants revealed that the AM-induced expression of SbtMl and the gene family members SbtM3 and SbtM4 is dependent on the common symbiosis pathway, whereas an independent pathway contributes to the activation of SbtS. We used the specific spatial expression patterns of SbtMl promoter ß-D-glucuronidase (GUS) fusions to isolate cis elements that confer AM responsiveness. A promoter deletion and substitution analysis defined two cis regions (region I and II) in the SbtMl promoter necessary for AM-induced GUS activity. 35S minimal promoter fusions revealed that either of the two regions is sufficient for AM responsiveness when tested in tandem repeat arrangement. Sequence-related regions were found in the promoters of AM-induced subtilase genes in Medicago truncatula and rice, consistent with an ancient origin of these elements predating the divergence of the angiosperms.