A novel delta-integration vector was developed to allow the sequential insertion of multiple cloned genes in the yeast Saccharomyces cerevisiae. To allow repetitive integrations, the reusable URA3 Blaster selection cassette was employed; the insertions (of CUP1p-lacZ in this study) were selected using the URA3 marker which was subsequently "popped" out by recombination between flanking direct repeats. Transformants contained only one new integrated copy after the loss of the URA3 marker, and subsequent transformations were effective for the sequential insertion of a series of genes (one at a time) into dispersed chromosomal delta sequences. The structural stability of the integrations was location-dependent (ranging from 75% to 100% after 50 generations in complex medium with or without gene expression), and the integrations (at least up to five) had no significant effects on the growth of the cells. In addition, beta-galactosidase specific activity levels varied linearly with integrated copy number. The repetitive, regulated nature of integration with this vector is not possible with traditional delta-integration or other homologous recombination methods, and is promising for fine-tuning cloned gene copy number and for the insertion of metabolic pathway genes.