The regulation of fatty acid biosynthesis by compounds that are required for growth of Bacillus thuringiensis was investigated using an vivo assay developed to measure fatty acid synthesis in germinating spores. A minimal glucose-ammonium-salts medium does not support growth even though previous radiorespirometric studies have shown B. thuringiensis to possess intact tricarboxylic acid and Embden-Meyerhof-Parnas pathways. Abundant growth does occur, however, when this medium is supplemented with either glutamate, aspartate, citrate, thiosulfate, cystine, or ethylenediaminetetraacetic acid. Cells held under nongrowing conditions incorporate acetate into fatty acids; fatty acid synthesis is stimulated by the compounds that permit growth. These alternate nutritional requirements are not manifestations of a vitamin or trace metal deficiency and do not reflect a chelation phenomenon. These results indicate a direct correlation between the capacity of these compounds to promote growth and to stimulate formation of fatty acids.