Thiolutin (Ia) and aureothricin (Ib) are shown to be acetamido and propionamido derivatives, respectively, of 3-amino-5-methylpyrrolin-4-ono-(4,3-d)-1,2-dithiole (IC). These similar yellow crystalline, sulfur-containing antibiotics are isolated from Streptomyces species and exhibit activity against fungi, ameboid parasites, Gram-positive, Gram-negative, and acid-fast bacteria. This paper reports studies elucidating Thiolutin's structure as the 3-acetamido derivative of IC, with aureothricin identified as the 3-propionamido derivative. Characterization includes infrared and ultraviolet spectral analysis, investigation of sulfur bonding (via comparison with dithiobenzoyl and sulfide reactions), hydrolysis of both antibiotics to the common product pyrrothine (IC, isolated as its hydrochloride salt) and regeneration via acylation with acetic/propionic anhydride, desulfurization of Thiolutin to desthiolutin (3-acetamido-1,5-dimethyl-2-pyrrolidone) (IIa) (synthesized to confirm structure), and degradation studies (alkaline hydrolysis yielding methylamine, ammonia, acetic acid; acid hydrolysis to pyrrothine). The unique pyrrolinonodithiole nucleus is defined, establishing the structural relationship between Thiolutin and aureothricin through chemical and synthetic evidence.