A number of antibiotics were tested as potential inhibitors of the purified trehalose-P synthase of Mycobacterium smegmatis. Of about 30 compounds tested, 4 (cathomycin, circulin, diumycin, and moenomycin) were active against this enzyme. Thus each of these compounds inhibited the formation of trehalose-P by the purified trehalose-P synthase when either UDP-glucose or GDP-glucose was used as the glucosyl donor. However, preincubation of the synthase with heparin, a polyanion activator of the enzyme when UDP-glucose is used as the substrate, prevented the inhibition by these various antibiotics. Fifty percent inhibition by diumycin and moenomycin occurred at a concentration of about 50 microg/ml (Ki of about 1 x 10(-5) M), but 50% inhibition by cathomycin and circulin required substantially higher concentrations (about 50 to 200 microg/ml). The inhibition by cathomycin, diumycin, and moenomycin was of the competitive type, whereas that by circulin was noncompetitive in nature. However, the inhibition was of a complex nature and the data suggest two different binding sites for these inhibitors. Photoaffinity labeling of the synthase with an azido-UDP-[32P]glucose probe was effectively blocked by diumycin, moenomycin, or cathomycin indicating that these inhibitors do interact at the substrate binding site. These antibiotics also inhibited the growth of M. smegmatis when added to cells innoculated into trypticase soy broth. The inhibition of growth was concentration-dependent and directly proportional to the size of the bacterial innoculum. These antibiotics, however, did not inhibit protein synthesis nor did they inhibit the incorporation of mannose into lipid-linked saccharides.