The biosynthesis of organof luorine compounds by Streptomyces cattleye NRRL 8057 was examined using 19F N M R spectroscopy. The organism produced 1.2 mM fluoroacetate and 0 5 mM 4-fluorothreonine as secondary metabolites when cultured for 28 d on a chemically defined medium containing 2 mM fluoride. Cell suspensions from batch cultures harvested at the growth maximum of 4 d were not capable of fluoride uptake or fluorometabolite biosynthesis, but by 6 d had developed an efficient fluoride-uptake system and biosynthesized the two f luorometabolites in almost equal proportions. As the harvest age increased, the proportion of f luoroacetate to 4-f luorothreonine formed by cell suspensions rose progressively so that 1640ld cells showed a ratio of 76:26 for the two compounds. Fluoride uptake and fluorometabolite production by cell suspensions were highly dependent on pH, with both processes showing a maximum rate a t pH 6 9 but declining rapidly at higher pH values. This decrease was particularly marked in the case of fluoroacetate biosynthesis which was barely detectable a t pH 7.5. Fluoroacetate and 4-f luorothreonine showed only low levels of interconversion by cell suspensions, suggesting that the carbon skeleton of neither was derived by metabolism of the other. The limited interconversion observed is explicable in terms of a small degree of biological def luorination occurring with each compound, followed by reincorporation of the resulting fluoride ion into the organic form by the active fluorinating system, a phenomenon also noted on incubation of cell suspensions with a number of other fluorinated biochemical intermediates. Cell suspensions were supplemented with a variety of amino acids and tricarboxylic acid cycle intermediates to determine the identity of the carbon substrate of the fluorinating system. No compound tested significantly increased the total amount of fluorometabolites formed or altered their relative proportions. However, in studies with 14C-labelled precursors, the highest level of incorporation into f luoroacetate by cell suspensions was recorded with [U-14C]glycolate, suggesting that this compound or an activated derivative may be the substrate for the fluorinating system in the biosynthesis of f luoroacetate.