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Fungi in the basidiomycetous ge nus Amanita owe their high mammalian toxicity to the bicyclic octapeptide amatoxins such as ∝-amanitin. Amatoxins and the related phallotoxins (such as the heptapeptide phalloidin) are encoded by members of the “MSDIN” gene family and synthesized on ribosomes as short (34 - 35 amino acid) proproteins. Anti-amanitin antibodies and confocal microscopy were used to determine the cellular and subcellular localization of amanitin accumulation in basidiocarps (mushrooms) of the Eastern North American destroying angel (Amanita bisporigera). Consistent with previous studies, amanitin is present throughout the basidiocarp (stipe, pileus, lamellae, trama, and universal veil), but it is present in only a subset of cells within these tissues. Restriction of amanitin to certain cells is especially marked in the hymenium. Several lines of evidence implicate a specific prolyl oligopeptidase, AbPOPB, in the initial processing of the amanitin and phallotoxin proproteins. The gene for AbPOPB is restricted taxonomically to the amatoxin-producing species of Amanita and is clustered in the genome with at least one expressed member of the MSDIN gene family. Immunologically, amanitin and AbPOPB show a high degree of co-localization, indicating that toxin biosynthesis and accumulation occur in the same cells and possibly in the same sub-cellular compartments.