The 1.2-kb DNA fragment of the Rickettsia conorii outer membrane protein B gene (OmpB(451-846)) was subcloned using site-specific PCR primers and expressed as six smaller fragments: OmpB(458-652), OmpB(595-744), OmpB(595-654), OmpB(645-692), OmpB(689-744), and OmpB(739-848). NCTC cells transfected with a mammalian expression vector expressing the fragments OmpB(689-744) and OmpB(739-848) stimulated immune anti-R. conorii CD8 T lymphocytes, suggesting the presence of CD8 T-lymphocyte-stimulating epitopes on these fragments. In order to further characterize the CD8 T-lymphocyte-stimulatory elements, CD8 T-lymphocyte epitopes on OmpB(689-744) and OmpB(739-848) were mapped by overlapping synthetic peptides. The ability of these synthetic peptides to stimulate immune CD8 T lymphocytes was determined by gamma interferon (IFN-gamma) production and cell proliferation after incubation with simian virus 40-transformed murine vascular endothelial cells in the presence of a 20 micro M solution of each synthetic peptide. Five synthetic peptides, SKGVNVDTV (OmpB(708-716)), ANVGSFVFN (OmpB(735-743)), IVSGTVGGQ (OmpB(749-757)), ANSTLQIGG (OmpB(789-797)), and IVEFVNTGP (OmpB(812-820)), induced secretion of IFN-gamma at significantly higher levels than the controls. Three of these five peptides, SKGVNVDTV (OmpB(708-716)), ANSTLQIGG (OmpB(789-797)), and IVEFVNTGP (OmpB(812-820)), also stimulated the proliferation of immune CD8 T lymphocytes. Significantly higher levels of specific cytotoxic T-lymphocyte killing were observed with the same three synthetic peptides, SKGVNVDTV (OmpB(708-716)), ANSTLQIGG (OmpB(789-797)), and IVEFVNTGP (OmpB(812-820)).