The role of protein-tyrosine kinases in bacterial polymyxin resistance was assessed by both genetic and biochemical approaches. Each of the two genes, wzc and etk, encoding protein-tyrosine kinases in Escherichia coli, was knocked out by using the PCR-based method of one-step inactivation of chromosomal genes, and the corresponding mutant strain was assayed in each case for resistance to different concentrations of polymyxin B by measuring the percentage of surviving cells. The resistance of a double knock-out wzc-etk-mutant was also analyzed and complementation experiments were performed by checking the effect of plasmid vectors expressing either Wzc or Etk. Our results concurred in showing that protein-kinase Wzc is not essential for polymyxin resistance, whereas protein-kinase Etk appears to play a key role in such antibiotic resistance. This newly found specific function of Etk reinforces the concept that protein-tyrosine kinases are involved in distinct facets of bacterial physiology.