A gene for chloroplast omega6 desaturase, which catalyzes the desaturation of monoenoic to dienoic acids in chloroplasts, was isolated from Chlamydomonas reinhardtii. We first performed reverse transcriptase-polymerase chain reaction with oligonucleotide primers corresponding to regions conserved among plastid omega6 desaturases of higher plants and delta12 desaturases of cyanobacteria, using C. reinhardtii poly(A)+ RNA. An amplified DNA fragment of 0.5 kb, containing a frame for a protein homologous to these desaturases, was used as a probe for screening cDNA and genomic DNA libraries of C. reinhardtii. The cDNA clone of 2.2 kb obtained contained an open reading frame encoding a protein of 424 amino acids with a putative molecular mass of 48.4 kDa, the amino acid sequence of which showed 46-51% homology to those of higher plant plastid omega6 and cyanobacterial delta12 desaturases. Introduction of the cloned genomic counterpart of this cDNA, designated as des6, into a Chlamydomonas mutant with defects in chloroplast omega6 desaturation and in the activities of photosystems I and II (PSI and PSII) complemented the desaturation mutation, indicating that the des6 gene codes for chloroplast omega6 desaturase. The complemented strains did not recover from the photosynthetic lesions, but showed lower PSII activity at 45 degrees C than the desaturation mutant, proving that the photosynthetic lesions in hf-9 are not caused by the desaturation mutation, and that the lowered unsaturation level of chloroplast lipids in the mutant is responsible for the expression at this high temperature of PSII activity, one of the thylakoid membrane functions.