We have chemically synthesized a DNA duplex of 757 base pairs which encodes the entire protein sequence of mature bacterio-opsin of Halobacterium halobium. The main aim of the synthesis was to facilitate site-specific mutagenesis in all parts of the gene by replacement of short restriction fragments by their counterparts containing the required nucleotide changes. Therefore, 30 unique restriction sites that are fairly evenly spaced were introduced in the synthetic DNA. A total of 28 oligonucleotides ranging in size from 21 to 69 nucleotides were synthesized corresponding to both strands. The entire gene was assembled from four synthetic fragments of 25, 268, 219, and 245 base pairs. The correctness of the nucleotide sequence was confirmed by sequencing the fragments as well as the complete gene. When expressed under the control of PL promoter in Escherichia coli, the synthetic and the native genes gave similar amounts of bacterio-opsin. Attempts to increase expression of the synthetic gene by introducing codons that are preferred in E. coli or by introduction of a synthetic transcription terminator were without significant effect. Work in this laboratory is focused on the structure-function studies of bacteriorhodopsin (bR),’ an integral membrane protein that serves as a light-driven proton pump in the extreme halophile, Halobacterium halobium. The approach involves extensive specific amino acid replacements in the protein by using methods of recombinant DNA. With this aim, we have reported on (a) the isolation and cloning of the gene for bR (1, 2), (b) the investigation and design of vectors for the expression of bacterio-opsin in Escherichia coli (3), and (c) methods for the purification and reconstitution of the expressed bO in a functional form (4).