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The sequences of the phosphoglycolate phosphatase (PGPase) gene Pgp1 and the 5′-upstream region from Chlamydomonas reinhardtii wildtype 2137 and the pgp1-1 mutant N142 that lacks the activity of PGPase (PGP1) were determined. The comparison revealed the alteration of a G to A at position 98 relative to the start codon. This destroyed the “GT” splice donor site at the beginning of the first intron of this gene, resulting in an extension of the first exon to 49 translatable codons followed by a stop codon, containing the codons corresponding to whole transit peptide for the chloroplast stroma and the first four N-terminal amino-acid residues of the PGP1 subunit. The comparison of the upstream nucleotide sequence of Pgp1 with those of 37 other genes including those involved in the CO2-concentrating mechanism and (or) photorespiration showed the high similarity of Pgp1 upstream to a periplasmic carbonic anhydrase gene Cah1; the motifs RAGGTCAGN8–9CCR and TTGGCAG were found only within the low-CO2 responsive genes, including Pgp1 and Cah1. GAN7CGNTTGGAAN2AG, TTGGAAGGAG, and CAGAGGTCAGN8CCG were found only with Pgp1 and Cah1, and ACGCTTGGCAGT and CATTACCAT were found only with Pgp1 and alanine aminotransferase gene Aat1. The possibility of functional PGPase isozyme(s) in C. reinhardtii is also discussed.