The ΔgalE, ΔpurA, and ΔaroA derivatives of avian septicemic Escherichia coli EC99 strain (O78 serogroup) were constructed with a suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis. The resultant isogenic mutants were stable and lacked approximately 45%, 36%, and 52% of the genes for galE, purA, and aroA, respectively. The ΔpurA and ΔaroA mutants did not grow on minimal medium, whereas the ΔgalE mutant grew on minimal medium but was sensitive to galactose-induced lysis. The reversion frequencies of all three mutants were <10⁻¹². The mutants were highly attenuated for virulence as determined by administration of approximately 10⁷ colony-forming units of each mutant to 1-day-old chicks by the subcutaneous route. Chickens were vaccinated with the mutants by spray (droplet size ∼20 μm) at 1 and 14 days of age to determine safety, immunogenicity, and efficacy. The mutants were found to be safe. Seven days after a second vaccination, immunoglobulin (Ig)Y antibodies to E. coli in serum and air sac washings were detected by enzyme-linked immunosorbent assay. In both serum and air sac washings, IgY antibodies were significantly higher in chickens vaccinated with the mutants as compared with phosphate-buffered saline–treated controls but were significantly lower compared with chickens that were vaccinated with the parent strain. In serum, but not in air sac washings, IgY antibodies were significantly lower in chickens vaccinated with the mutants compared with the parent strain. The vaccinated chickens were given infectious bronchitis virus intranasally at 17 days of age and were challenged with homologous (EC99 strain) or heterologous (O2 serogroup) E. coli 4 days later. Chickens that received wild-type EC99 strain or its mutant derivatives were protected from homologous but not from heterologous challenge. This study indicates that the ΔgalE, ΔpurA, and ΔaroA mutants are safe and moderately immunogenic but the protection conferred by the mutants is serogroup specific.