A revised U.S. Food and Drug Administration (FDA) method for the detection and isolation of Enterobacter sakazakii in powdered infant formula was developed based on real-time PCR technology complemented by culture isolation on chromogenic agars. A validation study was conducted to compare the revised FDA method to the reference FDA method. Casein and soy powdered infant formula inoculated with morphologically typical and atypical strains of E. sakazakii were analyzed. Valid results were obtained from 360 test portions and controls and showed that the revised FDA method is significantly better (P < 0.05) than the reference FDA method for the detection of typical E. sakazakii strains and the two methods are equivalent for the detection of atypical E. sakazakii strains.