In order to provide additional information for risk assessment of the fungicide metalaxyl, the main objectives were (1) to elucidate the interactions of metalaxyl with different human liver cytochrorne, P450 enzymes, (2) to tentitatively identify and (semi)quantify metabolites in vitro and (3) to identify human CYP enzymes responsible for metabolism. The mean inhibitory concentrations (IC50) for 7-pentoxyresorufin-O-dealkylation (CYP2B) and bupropion hydroxylation (2B6) were 48.9 and 41.7 mu M, respectively. The biotransformation reactions were hydroxylation, (di)demethylation and lactone formation. In human liver microsomes predominant metabolites were two hydroxymetalaxyl derivatives or atropisomers of one of the derivatives. On the basis of previous rat studies these could be N-(2-hydroxymethyl-6-methylphenyl)-N-(methoxyacetyl)alanine methyl ester and/or N-(2,6-dimethyl-5-hydroxyphenyl)-N-(methoxyacetyl)alanine methyl ester. The amounts of didemethylmetalaxyl N-(2,6-dimethylphenyl)N-(hydroxyacetyl)alanine and lactone 4-(2,6-dimethylphenyl)-3-methylmorpholine-2,5-dione were higher in homogenates than microsomes. The carcinogenic 2,6-dimethylaniline was not detected. Among the nine major human CYPs, CYP3A4 was the only one responsible for metalaxyl hydroxylation, while CYP2B6 was the major isoform responsible for (di)demethylation and lactone formation. (c) 2006 Elsevier B.V. All rights reserved.