Four serotypes of Salmonella enteritidis, Anatum ATCC 9270, Newbrunswick ATCC 1608, Oranienburg 200 E, and Pullorum RM, were studied to determine biological, chemical, or physical differences which might explain variations in Salmonella virulence as previously reported by McCullough and Eisele (J. Infect. Dis. 88:278-289, 1951; 89:259-265, 1951). These investigators found that serotype Pullorum was significantly less virulent than serotypes Newport, Derby, Barielly, Meleagridis and Anatum when fed to healthy humans. Results of our own experiments showed that serotype Pullorum RM had a generation time approximately twice that of serotype Anatum 9270. The volume of serotype Pullorum was approximately one-half the volume of the other serotypes used (Anatum 9270, Newbrunswick 1608, Oranienburg 200 E, Cubana 12007, and Meleagridis DR). The number of cells required to yield 1 g dry weight was substantially higher for serotype Pullorum RM than for serotypes Anatum 9270, Newbrunswick 1608, and Oranienburg 200 E. The yield of endotoxin per gram dry weight for serotype Pullorum RM averaged 22 mg/g, whereas yields of endotoxin for serotypes Anatum 9270, Newbrunswick 1608, and Oranienburg 200 E averaged 32 to 35 mg/g. The relative abundance of the four major fatty acids (measured by gas chromatography) also showed distinct differences among the serotypes. Pullorum RM contained less lauric and 3-hydroxymyristic acids and more myristic and palmitic acids than the other three serotypes. The identity of 3-hydroxymyristate was confirmed by mass spectroscopy. Serotype Pullorum RM required 10 times more lipopolysaccharides (endotoxin) to obtain a 50% lethal dose in mice than the other three serotypes. When the lipid part was separated from the polysaccharide and solubilized with bovine serum, the 50% lethal dose of serotype Pullorum RM was equal to that of the other three.