Rhizonin A, the main mycotoxin from Rhizopus microsporus, is a cyclic heptapeptide containing valine, allo-isoleucine, leucine, N-methylalanine, N-methyl-3-(3-furyl)alanine (molar ratio 2:1:1:1:2). Its amino-acid sequence was established by mass spectrometry; X-ray crystallography verified its structure, conformation, and relative configuration, while the absolute configuration was determined via high performance liquid chromatographic analysis of dansyl-derivatised amino-acids. Rhizonin A is the first mycotoxin reported from a fungus of the order Mucorales (class Phycomycetes). A highly toxinogenic R. microsporus strain (MRC 303), isolated from Mozambican groundnuts, was cultured on sterilized maize. Toxic principles were extracted with chloroform-methanol, and fractionation—guided by bioassays using day-old ducklings—involved solvent partitioning and chromatography on silica gel/aluminium oxide, yielding rhizonins A (0.01%) and B (0.00024%) relative to mouldered material. Rhizonin A has the molecular formula C42H65N7O9, with m.p. 243 °C, [α]D -25° (c 1.15, CHCl3), and spectral data (UV, IR, CD, ¹H/¹³C NMR) consistent with its structure. Three amino-acid residues are N-methylated. N,N-Dimethyl and N,N-bis(trideuteriomethyl) derivatives (lb, lc) were prepared via the Hakomori method, and amino-acid analysis of lb confirmed bismethylation sites. The molecular formula was validated by peak matching (m/z 811.486) and field desorption MS. Rhizonin B’s structure is based on amino-acid analysis and spectral comparisons. The rhizonins are the first natural products containing a 3-(3-furyl)alanine derivative, an amino-acid not previously known in peptides or as a natural product.