Streptomyces lusitanus var tetracyclini 106-T NCIB9500 is cultivated in an aq. nutrient medium contg. cornsteep liquor, CaCO3, starch or semi-hydrolyzate, ammonium salts, trace elements, peanut meal, and lard oil at a pH 6.7-6.8 for 140 hrs. at 26-8.degree. with aeration to obtain tetracycline in very high yields. There is no chlortetracycline produced. Thus, a prefermentor contg. 150 l. of medium is inoculated with starter culture and fermented at 26.degree. with agitation for 24 hrs. Then, a fermentor having a useful capacity of 6000 l. and contg. a medium of the following compn./l. of tap water: cornsteep liquor, CaCO3, starch, (NH4)2SO4, NH4Cl, MnSO4.bul.4H2O, CoCl2.bul.6H2O, ZnSO4, peanut meal, and lard oil 28, 14, 38, 5.7, 1.5, 0.05, 0.002, 0.05, 25, and 35 g., resp., is inoculated with the 24-hr. culture and fermented at 30.degree. for an addnl. 24 hrs., with aeration of 1.5 l./min./l. of broth. Then, the temp. is lowered to 26.degree. and the aeration increased to 4 l./min./l. of broth. After 140 hrs. of fermentation, 11.1 g. tetracycline/l. was obtained.