The shift to freshly cut leaves rather than scavenged dead vegetation as fungus-garden substrate was a major evolutionary transition in the attine ants that apparently allowed the Atta and Acromyrmex leafcutter ants to also evolve increased worker size dimorphism, multiple queen- mating, specific adaptations in the olfactory region of worker brains, and many other complex social traits. The transition also involved the specialization on a single species of fungal symbiont (Leucocoprinus gongylophorus), but comparative studies of the specific fungal adaptations that accompanied this transition have not been done. Such studies are important as the single specific fungal adaptation that can (almost) be seen with the bare eye (gongylidia; inflated hyphal tips that are preferentially eaten by the ants and their larvae) evolved earlier and therefore does not characterize the transition to leafcutting behaviour. Here we report the first large-scale comparative study on fungus garden enzyme profiles and show that various interesting changes can be documented. A more detailed analysis of laccase expression, an enzyme that is believed to oxidize phenols in defensive secondary plant compounds such as tannins, showed that this enzyme is exclusively found in the gardens of leaf-cutting ants, where it is significantly upregulated in the gongylidia. I’ll discuss the possible role of this enzyme and other fungal modifications in the evolution of the leafcutter ants and their non-leafcutting attine relatives.