A broad-host-range vector, pKT240, containing the structural gene (aph) for aminoglycoside phosphotransferase (APH), without promoter, has been constructed. Insertion of DNA fragments carrying promoters upstream of aph gene into the unique EcoRI site of this vector results in the expression of the aph gene and consequently the resistance of the host cells to streptomycin. The new vector has been used to show that the hybrid trp-lac (tac) promoter and the promoter of the lacIQ gene of Escherichia coli are active in Pseudomonas putida. Derivatives of pKT240 containing tac and lacIQ sequences may be used as wide-host-range expression vectors. Regulated overproduction of APH and catechol 2,3-oxygenase can be obtained with the aid of the new vectors in both E. coli and P. putida.