Primary infection by low pathogenic avian influenza (LPAI) predisposes for secondary infection by Escherichia coli in poultry, leading to significant economic losses. Future research in control of this ailment requires the establishment of a successful controlled challenge by avian influenza virus (AIV)/E. coli. Six groups of broilers (6 birds/group) were included for the standardisation of the controlled challenge by AIV/E. coli. Birds in groups 1, 2, 3, 4 and 5 received an intra-tracheal challenge of 0.5 ml of two haemagglutinating units of H9N2 virus at 20 days of age. At the age of 23 days, birds in group 1 received an intra-thoracic (right air sac)-E. coli challenge equivalent to 1.6 x 10 colony-forming units (cfu)/0.5 ml/bird, while birds in groups 2, 3, 4 and 5 received E. coli by the same route and in the following respective decreasing order of viable cells: 1.6 x 10(6), 1.6 x 10(5), 1.6 x 10(4) and 1.6 x 10(3); cfu. Birds in control group 6 were deprived of H9N2 and E. coli challenge. Results showed significant early mortality in group 1 that was challenged with the highest number of E. coli, in comparison to groups 2-6 (p<0.05); however, the average weight at 28 days of age was similar in surviving birds of groups 2-6 (p>0.05). The frequencies of four signs at 2 days and at 5 days post E. coli challenge (conjunctivitis, diarrhoea, ocular exudates and rales) in the surviving birds of groups 2-5 were most often higher than those observed in control group 6 (p<0.05). These four signs and five gross lesions (abdominal airsacculitis, left thoracic airsacculitis, pericarditis, right thoracic airsacculitis and tracheitis) had a decreasing pattern of frequency related to a decrease in the E. coli count used in the challenge.