Mutations in the five hrp and hrc genes in the hrpC operon of the phytopathogen Pseudomonas syringae pv. syringae 61 have different effects on bacterial interactions with host and nonhost plants. The hrcC gene within the hrpC operon encodes an outer membrane component of the Hrp secretion system that is conserved in all type III protein secretion systems and is required for most pathogenic phenotypes and for secretion of the HrpZ harpin to the bacterial milieu. The other four genes (in order), hrpF, hrpG, (hrcC), hrpT, and hrpV, appear to be unique to the group I hrp clusters found in certain phytopathogens (e.g., P. syringae and Erwinia amylovora) and are less well understood. We initiated an examination of their role in Hrp regulation and secretion by determining the effects of functionally nonpolar nptII cartridge insertions in each gene on the production and secretion of HrpZ, as determined by immunoblot analysis of cell fractions. P. syringae pv. syringae 61 hrpF, hrpG, and hrpT mutants were unable to secrete HrpZ, whereas the hrpV mutant overproduced and secreted the protein. This suggested that HrpV is a negative regulator of HrpZ production. Further immunoblot assays showed that the hrpV mutant produced higher levels of proteins encoded by all three of the major hrp operons tested-HrcJ (hrpZ operon), HrcC (hrpC operon), and HrcQB (hrpU operon)-and that constitutive expression of hrpV in trans abolished the production of each of these proteins. To determine the hierarchy of HrpV regulation in the P. syringae pv. syringae 61 positive regulatory cascade, which is composed of HrpRS (proteins homologous with sigma54-dependent promoter-enhancer-binding proteins) and HrpL (alternate sigma factor), we tested the ability of constitutively expressed hrpV to repress the activation of HrcJ production that normally accompanies constitutive expression of hrpL or hrpRS. No repression was observed, indicating that HrpV acts upstream of HrpRS in the cascade. The effect of HrpV levels on transcription of the hrpZ operon was determined by monitoring the levels of beta-glucuronidase produced by a hrpA'::uidA transcriptional fusion plasmid in different P. syringae pv. syringae 61 strains. The hrpV mutant produced higher levels of beta-glucuronidase than the wild type, a hrcU (type III secretion) mutant produced the same level as the wild type, and the strain constitutively expressing hrpV in trans produced low levels equivalent to that of a hrpS mutant. These results suggest that HrpF, HrpG, and HrpT are all components of the type III protein secretion system whereas HrpV is a negative regulator of transcription of the Hrp regulon.