Cell-free folate synthesizing extracts have been isolated from Escherichia coli, Mycobacterium lufu, Mycobacterium smegmatis (ATCC 607) and Mycobacterium leprae, and the inhibitory power of diaminodiphenylsulfone (DDS) in such cell-free systems on the synthesis of dihydropteroic acid has been determined. M. lufu and M. leprae extracts show very similar high sensitivities against DDS. Mode of action studies seem to indicate that the observed high sensitivity of M. lufu and M. leprae as compared to E. coli can be solely attributed to a high affinity for the dihydropteroic acid synthetase. A dihydropteroic acid analog formation where DDS is incorporated instead of p-aminobenzoic acid – as has been observed in E. coli – could not be detec