Glossophagine bats (Phyllostomidae, Glossophaginae) are specialized visitors to the flowers of several hundred species of neotropical plants. They are able to exploit flowers in hovering flight by imbibing nectar with a highly protrusile brush-tip tongue. As tongue extension is achieved by muscular and vasohydraulic mechanisms, its operational length can be inferred only from actively feeding animals. For this study, we measured maximum tongue extensions during nectar feeding in 9 species of glossophagine bats. We trained bats to feed from vertically oriented glass test tubes (9- and 15-mm inside diameter). The maximum depth of nectar drainage by a bat was recorded as maximum operational tongue length. Measured operational tongue lengths were in the range of the total body length of bats. The record length was 77 mm (in tubes with 15-mm inside diameter) in the 17-g flower specialist Choeronycteris mexicana. This compares with only 11–24 mm in the nonglossophagine frugivorous bat Carollia perspicillata, an opportunistic nectar feeder. The capacity for tongue extension proves the specialized status of neotropical glossophagines as flower visitors and clearly distinguishes them anatomically and ecologically from nonglossophagine nectar-feeding bats.