Abstract: An evaluation and optimization study of medium components for the production of the immunosuppressant compound rapamycin by Streptomyces hygroscopicus was addressed. Plackett-Burman experimental design was applied for screening of the most significant variables affecting production, where FeSO4, mannose, fructose and lysine-HCl were the most positive significant factors. In order to find out the combination among the most significant variables that brings maximum yield, Response Surface Methodology (RSM) was applied and the rapamycin yield increased to reach a theoretical value of 93 mg rapamycin per liter in the following medium (g L-1): Fructose, 90; mannose, 18.9; lysine-HCl, 16.8 and FeSO4, 0.1. Experimental verification of the polynomial model revealed a rapamycin yield of 95 mg L-1, which is an evidence of more than 98% accuracy of the model under the investigated conditions.