Dunaliella transformation using antisense and sense technology developed in this study will provide powerful tools for functional analysis and pathway-specific metabolic engineering in Dunaliella for industrial applications. Our aim was to investigate the potential of antisense expression and overexpression of a specific gene, the carotenoid biosynthesis-related (CBR) gene, in the microalgae Dunaliella. DNA amplified from sense and antisense vector constructs was used to transform Dunaliella tertiolecta. The Gateway vector for plant transformation was used to make an expression cassette, and the essential region for Dunaliella transformation was amplified by PCR and used for transformation. The transformation efficiency using a 3.2 kb PCR product was 130 transformants/μg DNA for both sense and antisense transformations. Among 200 BASTA-resistant colonies from the sense transformation and antisense transformation, separately, nine positive transformants for sense expression and five positive transformants for the antisense expression were obtained by genomic DNA PCR. The insertion was also verified by genomic Southern analysis. Among five positive sense transformants, one transformant was tested and verified for the overexpression of CBR-GFP fusion protein by Western blot analysis. One of the five antisense transformants showed almost complete inhibition of gene expression under light stress conditions (400 μmol photons m(-2) s(-1)) as determined by quantitative RT-PCR and Western blot analysis. Although there was no difference in the growth patterns or photosynthetic parameters between the wild type (including the vector control) and transformants, the zeaxanthin content of the antisense CBR mutant was lowered under light stress conditions. Thus, we show that the sense and antisense RNA technology can be easily and strategically used for the functional analysis of interesting gene in D. tertiolecta.