In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSF(ts1). The mutations that resulted in a temperature-sensitive phenotype in mSF(ts1) were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42 degrees C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 h of growth. In order to facilitate its utilization, the mSF(ts1) was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSF(ts1) for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.