AmpC enzymes can be differentiated from other extended-spectrum beta-lactamases by their ability to hydrolyse cephamycins as well as other extended-spectrum cephalosporins. The present study was designed to determine the occurrence of AmpC enzyme-harbouring Gram-negative clinical isolates in a tertiary care hospital in Delhi, India. Among the 135 clinical isolates of Gram-negative bacilli tested, 20.7% were found to harbour AmpC enzymes using a modified three-dimensional test. Inhibition of zone distortion in the presence of cloxacillin confirmed AmpC-harbouring isolates. Maximal incidence of AmpC producers was found among Acinetobacter spp. (42.8%) followed by Klebsiella pneumoniae isolates (33.3%). No AmpC-harbouring isolates revealed decreased susceptibility to cefoxitin. Therefore, Gram-negative bacilli showing resistance to any cephalosporin or aztreonam irrespective of cefoxitin susceptibility should be screened for the AmpC enzyme. The modified three-dimensional test is easy to carry out and can be applied as a phenotypic screening method for detection of AmpC-harbouring Gram-negative organisms. This is the first study to determine the occurrence of AmpC beta-lactamases from India.