The A. thaliana EST database was screened using consensus motifs derived from P450 families CYP52 and CYP4 catalyzing the omega-hydroxylation of fatty acids and alkanes in Candida and in mammals. One EST cDNA fragment was detected in this way and the corresponding full-length cDNA was cloned from a cDNA library of A. thaliana. This cDNA coded the first member of a new plant P450 family and was termed CYP86A1. The deduced peptide sequence showed highest homology with P450s from families 4 and 52. To confirm the catalytic function, CYP86A1 was expressed in a yeast overexpressing its own NADPH-P450 reductase. Efficient expression was evidenced by spectrophotometry, SDS-PAGE and catalytic activity. CYP86A1 was found to catalyze the omega-hydroxylation of saturated and unsaturated fatty acids with chain lengths from C12 to C18 but not of hexadecane. Genomic organization analyzed by Southern blot suggested a single gene encoding CYP86A1 in A. thaliana.