A lac-based alpha-complementation and expression system was developed for use in molecular cloning in Pseudomonas aeruginosa. A bacteriophage D3112-based mini-Dlac transposable element, containing the lacIq-regulated lacZ delta M15 gene next to a selectable marker, was constructed. Mixed D3112 lysates were used to transduce P. aeruginosa PAO1, and derivatives containing randomly inserted chromosomal copies of the mini-Dlac element were obtained. Transformation of the PAO1::mini-Dlac transductants with the broad-host-range vector, pUCP19, led to the formation of blue colonies on indicator medium in the presence of inducer. In contrast, transformants harboring the pUCP19 derivative pCDO, containing the catechol-2,3-dioxygenase (C23O)-encoding xylE gene under lac promoter control, were white on the same medium. Expression of xylE was tightly controlled by single-copy mini-Dlac-encoded lac repressor and in induced cultures was increased more than 100-fold over that observed in uninduced cultures. The usefulness of the system for molecular cloning in P. aeruginosa was demonstrated by ligating size-fractionated PAO1 chromosomal fragments into pUCP19, followed by transformation of the newly isolated PAO1::mini-Dlac host. All randomly chosen white colonies contained recombinant plasmids, with inserts of the correct size range, while blue colonies contained pUCP19 alone. The functionality of the system was also shown in another frequently studied strain, PA103.