Site-specific DNA integration allows predictable heterologous gene expression and circumvents extensive clone screening. Herein, the establishment of a Flipase (Flp)-mediated cassette exchange system in Sf9 insect cells for targeted gene integration is described. A tagging cassette harboring a reporter dsRed gene was randomly introduced into the cell genome after screening different transfection protocols. Single-copy integration clones were then co-transfected with both Flp-containing plasmid and an EGFP-containing targeting cassette. Successful cassette exchange was suggested by emergence of G418-resistant green colonies and confirmed by PCR analysis, showing the absence of the tagging cassette and single integration of the targeting cassette in the same locus. Upon cassette exchange, uniform EGFP expression between clones derived from the same integration site was obtained. Moreover, the resulting cell clones exhibited the expression properties of the parental cell line. EGFP production titers over 40 mg/L were of the same order of magnitude as those achieved through baculovirus infection. This Sf9 master cell line constitutes a versatile and re-usable platform to produce multiple recombinant proteins for fundamental and applied research. Biotechnol. Bioeng. 2012; 109: 2836–2844. © 2012 Wiley Periodicals, Inc.