PURPOSE: Bioluminescence imaging is a powerful tool for studying gene expression and cell migration in intact living organisms. However, production of bioluminescence by cells transfected to express luciferase can be limited by the rate of plasma membrane transport of its substrate D-luciferin. We sought to identify a plasma membrane transporter for D-luciferin that could be expressed alongside luciferase to increase transmembrane flux of its substrate and thereby increase light output. PROCEDURES: Luciferase-expressing cells were transfected with a lentivirus encoding the rat reno-hepatic organic anion transporter protein, Oatp1, which was identified as a potential transporter for D-luciferin. Light output was compared between cells expressing luciferase and those also expressing Oatp1. RESULTS: In two cell lines and in mouse xenografts, co-expression of Oatp1 with luciferase increased light output by several fold, following addition of luciferin. CONCLUSIONS: The increase in light output thus obtained will allow more sensitive detection of luciferase-expressing cells in vivo.