A rapid and efficient method to inactivate genes in Pseudomonas aeruginosa has been developed. It is based on pKnockout vectors which carry either a gentamicin or a streptomycin/spectinomycin resistance cassette allowing for selection in P. aeruginosa where these vectors do not replicate. A PCR fragment of the gene of interest carrying 5'- and 3'-truncations is cloned into a pKnockout vector, mobilized into P. aeruginosa, and subsequently integrated into the chromosomal copy of the target gene. The orientation of the fragment determines whether (i) the target gene is disrupted without blocking the transcription of downstream genes or (ii) the insertion exerts a polar effect thereby leading to inactivation of a whole operon.