Two new loci have been found to be clustered with five other genes for the nitrate assimilation pathway in the Chlamydomonas reinhardtii genome. One gene, located close to the 3'-end of the high-affinity nitrate transporter (HANT) gene Nrt 2; 2, corresponds to the nitrite reductase (NiR) structural gene Nii1. This is supported by a number of experimental findings: (i) NiR-deficient mutants have lost Nii1 gene expression; (ii) Nii1 mRNA accumulation is co-regulated with the expression of other structural genes of the nitrate assimilation pathway; (iii) nitrite (nitrate) utilization ability is recovered in the NiR mutants by functional complementation with a wild-type Nii1 gene; (iv) the elucidated NII1 amino acid sequence is highly similar to that of the cyanobacterial and higher-plant enzyme, and contains the predicted domains for plastidic ferredoxin-NiRs. Thus, the mutant phenotype and the mRNA sequence and expression of the Nii1 gene have been unequivocally related. Accumulation of mRNA for the second locus identified. Lde1 (light-dependent expression), was not regulated by nitrogen, but like nitrate-assimilation clustered genes, its expression was down-regulated in the dark.