It has been definitely established (Waksman, 1941; Waksman and Horning, 1942) that fungi capable of producing anti-bacterial substances are widely distributed in nature and are not limited to any one or more taxonomic groups. Such fungi can be readily isolated from soils, manures, composts, and dust by the use of appropriate bacteria-enriched agar media. Out of some 160 antagonistic fungi thus isolated, two organisms were selected for further study, because of their outstanding capacity to produce in culture media substances which inhibit in rather high dilutions the growth of various bacteria. These two organisms were Aspergillus fumigatus Fresenius, of which 16 strains were isolated from different soils and composts, and Aspergillus clavatus Desmazieres, represented by 3 isolations. The methods of testing the antibiotic action of the culture filtrates of these organisms have been described in detail elsewhere (Waksman and Homing, 1942). This paper deals specifically with the formation, concentration, and isolation of the antibiotic substances produced by these two fungi under different conditions of culture and nutrition. ASPERGILLUS FUMIGATUS When grown in simple synthetic media A. fumigatus produces an antibiotic substance, which has been designated as "fumigacin" (Waksman, Horning and Spencer, 1942). The medium used most extensively for this purpose was that of Czapek-Dox or a glucose-nitrate solution, which consists of 40 g. glucose, 3 g. NaNO3, 1 g. KH2PO4, 0.5 g. KCl, 0.5 g. MgSO4 7H20, 0.01 g. FeSO4.7H20, and 1000 ml. tap water.-The antibiotic substance was found to be produced in this medium during the early stage of growth of the organism: its presence could be demonstrated even within 3 days incubation at 28°C; it reached a maximum in 7-8 days, and then gradually disappeared on further incubation. The reduction in activity is due either to the destruction of the active substance or to its transformation into an inactive or at least a less active type of compound. In view of the recent announcement made by Oxford and Raistrick (1942) that fumigatin, a maroon-colored pigment produced by A. fumigatus, also has antibacterial properties, it was desirable to establish first the possible relation between the nature and formation of fumigatin and fumigacin, as well as their chemical and biological differences. This can be done by comparing the results on fumigacin obtained in this laboratory, with those on fumigatin reported from