Dentilisin (PrtP) is a virulence-related protease of Treponema denticola. We characterize prtP expression and dentilisin activity in biofilm and planktonic T. denticola cells under peptide limitation. Real-time PCR amplifying cDNA corresponding to internal fragments of individual genes and fragments spanning adjacent genes was used to assess gene expression. Biofilm and planktonic cell pools were incubated with either NOS broth or trypticase-diluted NOS broth for 12 h and assessed for gene expression. Peptidase activity was measured using a colorimetric-assay with SAAPFNA as substrate. prtP expression was greater in biofilm than planktonic cells; yet, biofilm cells expressed prtP consistently whereas planktonic cells increased transcription of prtP under peptide-limitation. Enzyme activity was also greater for biofilm than planktonic cells, but it remained consistent in both cell pools under peptide limitation. Similar patterns of expression were found for prcA and TDE0760, two genes upstream of prtP . Yet, these genes were expressed at significantly lower levels than prtP.