Capillary GC with flame ionization detection (FID) was used to determine the cellular fatty acid (CFA) profiles of six species in the new genus Cronobacter (Enterobacter sakazakii). The six different species are C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and C. genomospecies. For GC-FID analysis, whole cell fatty acid methyl esters (FAMEs) from cells cultured on brain heart infusion (BHI) agar at 35 degrees C for 24 h were obtained by saponification, methylation, and extraction into hexane-methyl tert-butyl ether. A data set for 57 strains of Cronobacter species was prepared using fatty acid profiles from two or three replicates prepared on different days. Major fatty acids of the Cronobacter strains evaluated in this study were straight-chain C12:0, C14:0, C16:0, and unsaturated C18:1, omega7c, summed C16:1 omega7c/C16:1 omega6c, and summed C14:0 3-OH/iso-C16:1, and C17:0 omega cyclo 7-8. The CFA profiles for the Cronobacter species are similar, but there are several fatty acids-C12:0, C14:0, C16:0, C18:1 omega7c, and summed C16:1 omega7c/ C16:1 omega6c--that differ significantly among these six species. Analysis of FAMEs from Cronobacter strains grown on BHI agar by a rapid GC-FID method is a sensitive procedure for the identification of these organisms, and this analytical method provides a procedure for the differentiation of strains from closely related Cronobacter species.