A series of isotope labelling experiments was carried out to investigate the biosynthesis of fluoroacetate and 4-fluorothreonine in resting cells of Streptomyces cattleya. Previous studies have shown that fluoroacetaldehyde is a precursor to both of these metabolites and the experiments were conducted to explore in greater detail the metabolic origin of fluoroacetaldehyde in S. cattleya. Ethanolamine and cysteamine are C-2 metabolites of serine and cysteine respectively and these two metabolites emerged as candidate precursors to fluoroacetaldehyde in S. cattleya. However feeding experiments with [1,1-H-2(2)]-ethanolamine and [1,1-H-2(2)]-cysteamine did not indicate incorporation into the fluorometabolites, suggesting that these compounds are not relevant precursors to fluoroacetaldehyde in S. cattleya. Upon feeding [2-H-2,2-O-18]-glycerol to resting cells of S. cattleya, the deuterium atom was not incorporated into 4-fluorothreonine, however the oxygen- 18 atom became incorporated into the carboxylate group of fluoroacetate and into the C(3)-O oxygen atom of 4-fluorothreonine. This observation indicates that there is an oxidation at C-2 of glycerol, but that the oxygen atom is formally retained from glycerol during the biosynthesis. In overview. the data suggest that fluoroacetaldehyde is derived from a C-3 glycolytic intermediate rather than a C-2 amino acid metabolite.