Dihydropteroate synthetase (DHPS) is specified by a substrain of Escherichia coli K12, ML1410. This enzyme activity is inhibited by sulfanilamides (Sa) and is known to be heat-stable, i.e., an Sa-sensitive normal enzyme. Another DHPS activity specified by E. coli ML1410 carrying drug resistance plasmids is Sa-resistant but heat-sensitive, i.e., an Sa-resistant enzyme. Most plasmids encoding single Sa or double (Sa. Tc or Sa. Sm) (Tc, tetracycline; Sm, streptomycin) resistance mediate the formation of this type of DHPS. Therefore, E. coli carrying these plasmids becomes diploid for DHPS, i.e., an Sa-resistant and an Sa-sensitive normal enzyme. The biochemical mechanism of Sa resistance mediated by plasmids encoding triple (Cm.Sm.Sa; Tc.Sm.Sa) and quadruple (Cm.Tc.Sm.Sa) resistance is not due to the formation of an altered DHPS but probably due to the decrease in permeation of the drug into the cell. The evolutionary process of the formation of Sa-resistance determinants on plasmids is discussed based on the presence of two types of Sa resistance mechanism.