Several asco-, basidio-, and zygomycetes isolated from an agricultural field were shown to be able to hydroxylate the phenylurea herbicide isoproturon [N-(4-isopropylphenyl)-N',N'-dimethylurea] to N-(4-(2-hydroxy-1-methylethyl)phenyl)-N',N'-dimethylurea and N-(4-(1-hydroxy-1-methylethyl)phenyl)-N',N'-dimethylurea. Bacterial metabolism of isoproturon has previously been shown to proceed by an initial demethylation to N-(4-isopropylphenyl)-N'-methylurea. In soils, however, hydroxylated metabolites have also been detected. In this study we identified fungi as organisms that potentially play a major role in the formation of these hydroxylated metabolites in soils treated with isoproturon. Isolates of Mortierella sp. strain Gr4, Phoma cf. eupyrena Gr61, and Alternaria sp. strain Gr174 hydroxylated isoproturon at the first position of the isopropyl side chain, yielding N-(4-(2-hydroxy-1-methylethyl)phenyl)-N',N'-dimethylurea, while Mucor sp. strain Gr22 hydroxylated the molecule at the second position, yielding N-(4-(1-hydroxy-1-methylethyl)phenyl)-N',N'-dimethylurea. Hydroxylation was the dominant mode of isoproturon transformation in these fungi, although some cultures also produced traces of the N-demethylated metabolite N-(4-isopropylphenyl)-N'-methylurea. A basidiomycete isolate produced a mixture of the two hydroxylated and N-demethylated metabolites at low concentrations. Clonostachys sp. strain Gr141 and putative Tetracladium sp. strain Gr57 did not hydroxylate isoproturon but N demethylated the compound to a minor extent. Mortierella sp. strain Gr4 also produced N-(4-(2-hydroxy-1-methylethyl)phenyl)-N'-methylurea, which is the product resulting from combined N demethylation and hydroxylation.