A new small plasmid vector (pKSS) for the direct selection of insert-containing plasmid clones is presented. The selection strategy is based on the acquired sensitivity of Escherichia coli cells to p-chloro-phenylalanine (p-Cl-Phe) if they carry a pheS allele encoding a phenylalanyl-tRNA synthetase alpha subunit with relaxed substrate specificity. This pheS allele is present on pKSS. Insertion into, or replacement of, the plasmidial pheS gene by a cloned fragment enables transformed pheS wild-type cells to survive on agar plates containing p-Cl-Phe plus ampicillin. This host strain-independent positive selection of recombinant clones proved to be highly efficient (> 99%) and did not require purification of the vector fragment prior to cloning. The high-copy-number vector pKSS offers a multitude of restriction sites and all of the features for analysis of cloned fragments that stem from the cloning vector pBluescript (Stratagene, La Jolla, CA, USA). Thus, pKSS represents a valuable alternative to previously reported positive-selection vectors; it should prove particularly useful for cloning when expecting a high fraction of cells transformed with non-recombinant vector, and for construction of DNA libraries.