Pseudomonic acid A (1) has been the dominant commercial pseudomonate antibiotic produced by Pseudomonas fluorescens. In specific shaken flask conditions initial fermentation accumulation of 1 is followed by preferential accumulation of the 8-hydroxy derivative, pseudomonic acid B (2). Biosynthetic probing with a pulse of [1-14C] acetate or L-[methyl-14C] methionine at early, mid and late stages of the fermentation gave relative patterns of radioactivity in 1 and 2 that are inconsistent with an assumption that 2 arises by oxidation of 1, or that 1 is formed by reduction of 2. Since [methyl-14C] methionine only labels carbons in the 12-carbon part of the pseudomonate molecule that is thought to be an early biosynthetic moiety, the evidence from radiolabelling experiments implies that preferential early oxidation of this biosynthetic intermediate causes the pathway diversion to accumulate 2 instead of 1.