The Ty3 retrotransposon of Saccharomyces cerevisiae was employed for the site-specific integration of heterologous genes into the yeast genome. A GAL-regulated promoter allowed induction of the retrotransposition process, and a bacterial neo(r) gene inserted in the Ty3 element was used as a selectable model heterologous gene. The frequency of transposition of this neo(r)-marked element was found to be comparable to that of an unmarked element. Th ree amplification systems were constructed; the systems varied with respect to the location and number of the GAL-regulated helper and neo(r)-marked Ty3 elements. For all three systems, neo(r) integrations were readily selected with a maximum of two insertions obtained per round of amplification. A sequential amplification strategy was effective for further increasing the number of integrated cloned genes, and families of strains varying by only one neo(r) insertion were easily obtained. Resistance to the antibiotic G418 correlated well with the number of integrated neo(r) genes, and Northern blots verified the relationship between cloned gene number (up to four) and neo(r) expression. Structural stability of the integrated genes was also demonstrated. By controlling the number of rounds of amplification and the level of G418 selection, precise numbers of integrated heterologous genes could be obtained. Because the amplification process can be repeated using different cloned genes inserted in the Ty3 element, these results demonstrate the potential of retrotransposition for the regulated integration of a series of different genes at nondeleterious chromosomal locations.