The leaf-cutting ants Acromyrmex octospinosus live in symbiosis with a fungus they cultivate on leaf fragments. The symbiote forms characteristic hyphal apical swellings called “gongylidia,” which are consumed in preference to the mycelium by the ants. However, the ants also ingest fluids derived from the plant leaves they handle to make the culture substrate. The 4-desmethylsterols of the ants were essentially (83%–94% of total 4-desmethylsterols) Δ5,7-sterols which were identified as ergosta-5,7,24(28)-trien-3β-ol, ergosta-5,7-dien-3β-ol, and ergosterol. These Δ5,7-sterols were all found in the gongylidia, whereas ergosterol mainly was found in the mycelium. Some Δ5-sterols were detected in the whole body, as well as in the expressed rectal fluid of the ants which partly contains the nonabsorbed material. However, the rectal fluid, as compared to the whole body, contained much higher amounts of Δ5-sterols originating mainly from the plant leaves. Leaf-cutting ants preferentially retained Δ5,7-sterols originating from the specialized gongylidia formed by the fungal symbiote, from a mixed diet of fungal and plant leaf sterols. © 1992 Wiley-Liss, Inc.