Because iron acquisition is essential to the survival of invasive strains of Escherichia coli, the frequency of two potential iron acquisition systems, aerobactin and hemolysin production, were compared in E. coli isolated from human blood (n = 95), urine (n = 100), and stool (n = 50). By phenotypic and genotypic methods, the prevalence of hemolysin production was 22% in bacteremic, 38% in urinary, and 22% in fecal isolates of E. coli. Aerobactin production was detected in 76% of blood and in 73% of urinary isolates but in only 52% of fecal isolates (P less than .01). A reciprocal relationship was found in blood isolates between aerobactin and hemolysin; the majority of blood isolates (55%) that lacked aerobactin were hemolytic, whereas only 14% of blood isolates that expressed aerobactin were hemolytic (P less than .0001). Aerobactin may be the principal mechanism of iron acquisition in extraintestinal isolates of E. coli, and hemolysin may serve as an alternative mechanism in the absence of aerobactin genes.