The work applied a transgene expression method based on the replacement of an inactive rbc L gene as the selection marker in Chlamydomonas reinhardtii chloroplasts. The native rbc L gene in strain CC2653 has a point mutation that causes early translation termination, thus resulting in a photosynthesis mutant. Recovery of rbc L function for selection is offered along with the heterologous expression of the alcohol dehydrogenase ADH1 gene from Saccharomyces cerevisiae in the Chlamydomonas chloroplast. The Cr Cp ADH1 gene was inserted via double homologous recombination in the psa B-rbc L chloroplast intergenic region of recipient strain CC2653, using the psa B and rbc L gene sequences for the double homologous recombination. This transformation conferred a functional rbc L gene and expression of the Cr Cp ADH1 transgene in the recipient strain. This method alleviated the need to use antibiotics for selection, resulting in a negligible number of false positives during screening, and attaining a transformation efficiency greater than 90%. The approach also ensured segregation of chloroplast DNA copies, so as to achieve homoplasmy of the transformant chloroplast DNA, with a concomitant elimination of recipient strain Cp DNA. High levels of steady-state Cr Cp ADH1 transcripts were detected in the homoplasmic transformants. However, Cr Cp ADH1 protein levels were attenuated under continuous illumination growth conditions due to oxygen accumulation in the cells. Under conditions of low oxygen partial pressure, or anoxia, accumulation of Cr Cp ADH1 protein in the cells and ethanol in the growth medium was observed. A metabolic pathway for ethanol production is proposed in Chlamydomonas, mediated by the chloroplast-localized Cr Cp ADH1 transgenic enzyme. [ABSTRACT FROM AUTHOR]