The hypothetical schemes proposed for the biosynthesis of unsaturated mycolic acids (R1-CH(OH)-CH(R2)-COOH) of Mycobacteria cell walls were experimentally tested by using cell-free extracts either of Mycobacterium aurum or of Mycobacterium smegmatis which produce two kinds of unsaturated mycolic acids (mono and dialkene), [1-14C]acetate being the precursor. Examination of specific radioactivities, in the presence or in the absence of isoniazid, an antituberculous drug inhibiting mycolic acid synthesis, showed that saturated C22 and C24 acids play a role as precursors of two distinct parts of the mycolic acids. Moreover, determination of labelling distribution into mycolic acid fragments obtained by oxidative and pyrolytic cleavages showed first that the side chain R2 and the methyl end R1 both have these C22 and C24 saturated fatty acids as common precursors. Secondly, it is thought that the fragments located between the methyl end R1 and the side chain R2 mainly result from elongation steps (one or two successive additions of seven or eight C2 units according to the mycolic acid type) and a biosynthetic model is proposed for unsaturated mycolic acids extending the published models and illustrating the missing step in monoalkene formation.