We have constructed a yeast integration vector targeted to chromosomal Ty δ sequences and used it to create Saccharomycescerevisiae strains with stable tandem integrations ranging from 1 to 30 vector copies. The vector carries the bacterial NEO gene, allowing copy number to be tuned by varying G418 resistance, which generally increases with copy number as determined by quantitative Southern blot. Tandem integration into a single site is most commonly observed, but single-copy and two-site integration is also observed. Bovine pancreatic trypsin inhibitor was constitutively expressed and secreted using the NEO-based δ vector, and secretion levels were 2−10-fold improved relative to commonly used 2μ multicopy yeast plasmids. The NEO-based Ty δ vector is a powerful tool for stable heterologous protein expression and secretion in yeast.