The biosynthetic origin of the pyrrolidone ring in pramanicin 1 (an inhibitor of Cryptococcus neoformans isolated from Stagonospora sp. ATCC 74235) was unclear, with two potential pathways proposed: one via proline/glutamate and another via a tetramic acid intermediate with serine. To determine the operational pathway, we performed preliminary labelling experiments using labelled acetates and serine. Incorporation of these labelled precursors into pramanicin 1 showed that its carbon skeleton is derived from eight acetate units and a serine residue, implying that the biosynthesis of pramanicin 1 proceeds via an acyl–tetramic acid intermediate 3.