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An especially interesting question is "How many species of bats can be found simultaneously in the same cave?" This information is surprisingly rare in the literature, mainly in the Neotropics. The aim of this study was to sample bats in Gruta do Limoeiro cave, Municipality of Castelo, State of Espírito Santo, Brazil after a 53-year interval of the first survey, by the naturalist Augusto Ruschi. Four surveys were conducted in 2005 and 2006, capturing bats with mist-nets and actively exploring the cave. We recorded eleven species of three families, Phyllostomidae, Vespertilionidae and Molossidae, making Gruta do Limoeiro cave one of the most diverse caves in the World for bats. Of the 14 species found by Ruschi, seven were still present, and four species were added. The seven species lost from Ruschi's list are mainly Emballonuridae and some Phyllostomidae, probably due to landscape changes. We recommend the long-term monitoring of the Gruta do Limoeiro cave to understand the loss in diversity, and consequently in ecosystem services. [ABSTRACT FROM AUTHOR]