The feasibility of two strategies for transgene pyramiding using Agrobacterium -mediated transformation was investigated to develop a transgenic potato ( Solanum tuberosum L. cv. Iwa) with resistance to potato tuber moth (PTM) ( Phthorimaea operculella (Zeller)). In the first approach, cry 1Ac9 and cry 9Aa2 genes were introduced simultaneously using a kanamycin ( npt II) selectable marker gene. The second approach involved the sequential introduction (re-transformation) of a cry 1Ac9 gene, using a hygromycin resistance ( hpt ) selectable marker gene, into an existing line transgenic for a cry 9Aa2 gene and a kanamycin resistance ( npt II) selectable marker gene. Multiplex polymerase chain reaction (PCR) confirmed the presence of the specific selectable marker gene and both cry genes in all regenerated lines. The relative steady-state level of the cry gene transcripts in leaves was quantified in all regenerated lines by real-time PCR analysis. Re-transformation proved to be a flexible approach to effectively pyramid genes for PTM resistance in potato, since it allowed the second gene to be added to a line that was previously identified as having a high level of resistance. Larval growth of PTM was significantly inhibited on excised greenhouse-grown leaves in all transgenic lines, although no lines expressing both cry genes exhibited any greater resistance to PTM larvae over that previously observed for the individual genes. It is anticipated that these lines will permit more durable resistance by delaying the opportunities for PTM adaptation to the individual cry genes.