Fruiting bodies from the Agaricus genus have been found to contain non-toxic arsenobetaine (AB) as a major compound.It is unknown whether AB is formed during the vegetative or reproductive life stages of the fungus,or by the surrounding microbial community,but AB’s structural similarity to glycine betaine has led to the hypothesis that AB may be adventitiously accumulated as an osmolyte.To investigate the potential formation of AB during the reproductive life stage of Agaricus species,growth substrate and fungi were collected during the commercial growth of Agaricus bisporus and analyzed for arsenic speciation using HPLC-ICP-MS.AB was found to be the major arsenic compound in the fungus at the earliest growth stage of fruiting (the primordium).The growth substrate mainly contained arsenate (As(Ⅴ)).The distribution of arsenic in an A.bisporus primordium grown on As(Ⅴ) treated substrate,and in a mature Agaricus campestris fruitingbody collected from arsenic contaminated mine tailings,was mapped using two dimensional XAS imaging.The primordium and stalk of the mature fruiting body were both found to be growing around pockets of substrate material containing higher As concentrations,and AB was found exclusively in the fungal tissues.In the mature A.campestris the highest proportion of AB was found in the cap,supporting the AB as an osmolyte hypothesis.The results have allowed us to pinpoint the fungus life stage at which AB formation takes place,namely reproduction,which provides a direction for further research.