A newly isolated Pseudomonas plasmid coding for detoxification of the herbicide paraquat (Pq(r)) was characterized. An EcoR1-generated fragment derived from the plasmid carrying the Pq(r) determinant was cloned into Escherichia coli. Subsequent subclonings, followed by exonuclease III-mediated deletion analysis, localized the Pq(r) gene(s) to a 1.8-kb segment within a 4.2 Pst1 subfragment. The cloning and apparent expression of the Pq(r) gene(s) in E. coli will enable its structural organization and function to be analyzed in detail.