Previously published reports have noted biochemical reactions atypical of Salmonella among the Ames tester strains of Salmonella typhimurium, and an inability to assign the strains to a specific Salmonella O (heat-stable cell wall) antigen group. We studied the biochemistry and serology of strains TA97, 98, 100, 102, 104, 1535, 1537, and 1538 in an attempt to develop a protocol to correctly speciate the strains. Biochemical reactions of all eight strains using standard media supplemented with histidine and biotin were consistent with those of the genus Salmonella. Strains TA100, 104, and 1535 were assigned to Salmonella O groups using bacteria treated with hot ethanol (White schema). H (flagellar) antigen assignments were performed successfully with seven of the eight strains. Two H antigen assignments required the use of the Craigie tube test for selection of motile revertants. Combining our biochemical and serological results obtained by this protocol, we were able to correctly speciate TA100, 104, and 1535 as Salmonella typhimurium. Our results demonstrate that representatives of the tester strains can be correctly speciated provided that procedures are followed that allow for the unusual nutrient requirements, the deep rough cell wall mutation, and the variably deficient motility of these organisms.