The ticks examined were in the early middle to middle pre-moulting period corresponding to the nymph-pharate adult transitional phase. Although Borrelia burgdorferi could be found intracellularly within Malpighian tubule, ovarian and syncytial muscular tissues, the spirochaete was predominantly located in the extracellular sites associated with different organs. A large number of borreliae were observed in the very narrow endoperitrophic space resulting from the peritrophic membrane atrophy in systemically infected nymphs at day 15 after a non-infectious blood meal. This observation indicates that some spirochaetes in the midgut lumen of unfed nymphs are enveloped within the endoperitrophic space once the peritrophic membrane occurs and/or condenses and survive there at least until ticks begin to enter pharate stage of the adult. The absence of B. burgdorferi from the ectoperitrophic space in systemically infected early middle to middle moulting nymphs suggests that spirochaetes persisting in this space may become systemic thereafter. The detection of borreliae only in the narrow endoperitrophic space of a male nymph at day 15 after repletion indicates that all pre-feeding midgut spirochaetes may be enclosed in this space when the peritrophic membrane appears and/or condenses, and the peritrophic membrane may become a barrier preventing spirochaetes from penetrating the midgut epithelium.