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We used bioassays to determine the sex pheromone production site in the female American cockroach (Periplaneta americana). Bioassayed extracts from various body parts aroused sexual responses from unmated males. However, the extract of hindgut (including both colon and rectum) induced strong responses. Colon extract stimulated the strongest sexual response. To compare the sex pheromone contents of atrial glands, pygidium, rectum, and colon, we recorded the EAG responses of male antennae to these extracts. Among the four tissues, colon extract caused the strongest EAG response. From gas chromatographic (GC) analysis, the quantities of periplanone-A (PA) and periplanone-B (PB) (the main sex pheromone components in female American cockroach) were 0.34 and 8.31 ng, respectively, in the colon of a virgin female. The ratio of PA to PB was ca. 1:24. The present results are different from those reported in a previous study, in which it was concluded that atrial glands are the sites of sex pheromone production in P. americana, and from GC analysis the quantity of PB was estimated to be 60 ng/female. There was no difference in sexual behaviors induced by colon and atrial glands of calling virgin females. It is unclear whether the colon is the sole sex pheromone source in the female American cockroach.