When grown photoautotrophically at air levels of CO 2, Chlamydomonas reinhardii possesses a system involving active transport of inorganic carbon which increases the intracellular CO 2 concentration considerably above ambient, thereby stimulating photosynthetic CO 2 assimilation. In previous investigations, two mutant strains of this unicellular green alga deficient in some component of this CO2-concentrating system were recovered as strains requiring high levels of CO 2 to support photoautotrophic growth. One of the mutants, ca-lq2-1C, is a leaky (nonstringent) CO2-requiring strain deficient in carbonic anhydrase (EC 4.2.1.1) activity, while the other, pmp-l-16-5K, is a stringent CO2-requiring strain deficient in inorganic carbon transport. In the present study a double mutant (ca prop) was constructed to investigate the physiological and biochemical interaction of the two mutations. The two mutations are unlinked and inherited in a Mendelian fashion. The double mutant was found to have a leaky CO2-requiring phenotype, indicating that the mutation ca-1 overcomes the stringent CO2-requirement conferred by the mutation prop-1. Several physiological characteristics of the double mutant were very similar to the carbonic-anhydrase-deficient mutant, including high CO2 compensation concentration, photosynthetic CO 2 response curve, and deficiency of carbonic-anhydrase activity. However, the labeling pattern of metabolites during photosynthesis in ~4CO2 was more like that of the bicarbonatetransport-deficient mutant, and accumulation of internal inorganic carbon was intermediate between that of the two original mutants. These data indicate a previously unsuspected complexity in the Chlamydomonas C02-concentrating system.