SR31747 is a novel agent that elicits immunosuppressive and anti-inflammatory effects. This drug was shown to inhibit Delta8-Delta7 sterol isomerase in yeast. To test whether this enzyme could also be an SR31747 target in mammals, the binding, antiproliferative and sterol biosynthesis inhibitory properties of various drugs were studied in recombinant sterol isomerase-producing yeast cells. Our results clearly show that SR31747 is a high affinity ligand of recombinant mammalian sterol isomerase (Kd = 1 nM). Tridemorph, a sterol biosynthesis inhibitor that is widely used in agriculture as an antifungal agent, is also a powerful inhibitor of murine and human sterol isomerases (IC50 value in the nanomolar range). Some drugs, like cis-flupentixol, trifluoperazine, 7-ketocholestanol and tamoxifen, inhibit SR31747 binding only with the mammalian enzymes, whereas other drugs, like haloperidol and fenpropimorph, are much more effective with the yeast enzyme than with the mammalian ones. Emopamil, a high affinity ligand of human sterol isomerase, is inefficient in inhibiting SR31747 binding to its mammalian target, suggesting that the SR31747 and emopamil binding sites on mammalian sterol isomerase do not overlap. In contrast, SR31747 binding inhibition by tamoxifen is very efficient and competitive (IC50 value in the nanomolar range), indicating that mammalian sterol isomerase contains a so-called antiestrogen binding site. Tamoxifen is found to selectively inhibit sterol biosynthesis at the sterol isomerase step in the cells that are producing the mammalian enzyme in place of their own sterol isomerase. Finally, we also show that tridemorph, a sterol biosynthesis inhibitor widely used in agriculture as an antifungal agent, is not selective of yeast Delta8-Delta7 sterol isomerase but is also highly efficient against murine Delta8-Delta7 sterol isomerase or human Delta8-Delta7 sterol isomerase. This observation contrasts with our already published results showing that fenpropimorph, another sterol isomerase inhibitor used in agriculture, is only poorly efficient against the mammalian enzymes.