The mechanisms mediating the association between Escherichia coli and specific hosts are unknown. This study investigates the hypothesis that the host-specific associations of E. coli strains are mediated in part by differences in chemotaxis. To test this hypothesis, chemotactic responses of E. coli strains isolated from different host groups (carnivores, herbivores, and omnivores) were tested with various attractants. In low-density agar chemotaxis assays, the average motility of E. coli in response to aspartate, serine, and ribose among the different groups was not significantly different; however, strains from carnivores responded significantly more to aspartate, relative to their responses to serine, in comparison with strains from herbivores, which responded equally or better to serine than to aspartate. The relatively greater chemotactic response of strains from carnivores to aspartate than to serine was confirmed in a subset of strains by capillary chemotaxis assay. Differences in responses to serine and aspartate were not due to growth differences, as determined by comparison of 24 h growth curves with glycerol, aspartate, and serine carbon sources. The differences in chemotactic behavior of E. coli strains isolated from herbivores and carnivores support the hypothesis that host-specific associations of E. coli strains are mediated in part by differences in chemotactic behavior.