Glyphosate (Roundup) is an herbicide used extensively worldwide which acts as an inhibitor of 5'enolpyruvylshikimate-3-phosphate synthase and for which transgenic herbicide resistant plants have been developed. Here we report for the first time that glyphosate is an inhibitor of cytochrome P450 using a functional expression system for Thlaspi arvensae CYP71B1 in Escherichia coli. CYP71B1 was fused to the soluble domain of a plant cytochrome P450 reductase (CPR) from Catharanthus roseus. CYP71B1 could obtain reducing equivalents in this fusion construct and metabolised the polycyclic aromatic hydrocarbon, benzo(a)pyrene. The fusion protein retained normal spectral characteristics having a Soret peak at 448 nm in the reduced carbon monoxide difference spectrum. Addition of the herbicide resulted in a Type II spectrum indicative of binding via the nitrogen group to haem as a sixth ligand. A Ks of 60 microM was observed and an IC50 of 12 microM was observed for glyphosate inhibition of CYP71B1 activity. The implications of these results are discussed.