From fermentations of Tolypocladium niveum supplemented with D-threonine, a novel natural cyclosporin, [Melle4]cyclosporin, was isolated. Its structural elucidation is based on amino acid analysis and spectroscopic data; the amino acid sequence was deduced from two-dimensional NMR investigations applied to the iso-derivative of [Melle4]cyclosporin which, in contrast to the natural product, is present as one homogenous conformation in solution. We show that one of the four N-methyl-L-leucine units of cyclosporin A, namely that in position 4, is replaced by N-methyl-L-isoleucine. The putative mechanism by which D-threonine induces in vivo biosynthesis of [Melle4]cyclosporin is discussed. In vitro biosynthesis of [Melle4]cyclosporin was achieved using the previously described enzymatic system [Lawen and Traber (1993) J Biol Chem 268: 20452-20465], thereby demonstrating the high affinity of cyclosporin synthetase for isoleucine in position 4. In a long series of cyclosporins obtained by in vitro and in vivo biosynthesis, [Melle4]cyclosporin represents the first example that is devoid of immunosuppressive efficacy while retaining strong binding to cyclophilin. It exerts potent in vitro anti-HIV-1 activity.