Thiolactomycin inhibited fatty acid synthesis in isolated pea chloroplasts. Using [1- 1 4 C]acetate as a precursor, an I 5 0 value of ca 20 μM was obtained for a (R,S) mixture of thiolactomycin. The antibiotic inhibited the β-ketoacyl-ACP synthase (condensing enzyme: KAS) and the acetyl-CoA:ACP (ACAT) reactions. An examination of the relative inhibition of the three condensing enzymes revealed that their order of sensitivity to thiolactomycin was KAS II > KAS I > KAS III.