Abstract Bacillus licheniformis 9259 has been shown to synthesize two C16 polyunsaturated fatty acids from endogenous or added palmitate at 20°, but not at 35°. Ready conversion of added palmitic acid labeled with 14C or 3H to these fatty acids by cultures of B. licheniformis incubated at 20° permitted the isolation of sufficient amounts of material for the determination of structure and label distribution by physical and chemical methods. The major polyunsaturated component was shown to be 5,10-hexadecadienoic acid while a minor component had the 7,10-double bond configuration. It was further shown that the biosynthesis of the major dienoic acid resulted from the cooperative action of two distinct desaturation systems. One system, which was present at both 35 and 20° desaturated palmitic acid to 10-hexadecenoic acid. A second system, active only at the lower temperature, resulted in the conversion of palmitic acid to 5-hexadecenoic acid. This system, which had previously been studied in Bacillus megaterium, is apparently not present at 35° but is rapidly induced at 20°. Utilizing 3H-labeled 10-hexadecenoic and 5-hexadecenoic acids which were prepared biosynthetically, it was possible to show that, at 20°, either substrate could serve as a precursor for the biosynthesis of 5,10-hexadecadienoic acid by B. licheniformis. Indeed, they were both more effective in this regard than palmitic acid added in similar concentrations. Although added palmitic acid is also converted to 7,10-hexadecadienoic acid at 20°, there was no evidence for the formation of the monoene, 7-hexadecenoic acid at either 35 or 20°. Presumably, the substrate for Δ7 desaturation is 10-hexadecenoic acid. The finding that B. licheniformis can carry out the conversion of palmitate to 5,10- and 7,10-hexadecadienoic acids apparently represents the first unequivocal indication of polyunsaturated fatty acid biosynthesis de novo in bacteria.