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The Neotropical Heliconius butterflies are well known to supplement their nectar diet by active pollen collecting. They extract proteins and free amino acids from pollen grains, exhibiting a particular behavior that involves the use of a fluid of uncertain origin. It has been assumed that this fluid is either regurgitated nectar or saliva, because for anatomical reasons a butterfly is able to release only these two fluids through its proboscis. In an experimental approach, 27 individuals of Heliconius melpomene (L.) were given red-dyed sugar solution and subsequently we observed whether the fluid used in pollen feeding was dyed or not dyed. Because regurgitated nectar should contain sugar, fluid samples were taken from the proboscis of butterflies from natural populations in Costa Rica. Samples of 44 individuals from seven species were tested for the presence of fructose and glucose with the aid of aniline phthalate. This study is the first detailed investigation of the origin of the fluid used by Heliconius butterflies in pollen feeding. The results are discussed in terms of already existing hints in literature concerning the true nature of that fluid. [ABSTRACT FROM AUTHOR]