The properties and role of the enzyme phosphoglycolate phosphatase in the cyanobacterium Coccochloris peniocystis have been investigated. Phosphoglycolate phosphatase was purified 92-fold and had a native molecular mass of approximately 56 kilodaltons. The enzyme demonstrated a broad pH optimum of pH 5.0 to 7.5 and showed a relatively low apparent affinity for substrate (K(m) = 222 micromolar) when compared to that from higher plants. The enzyme required both an anion and divalent cation for activity. Mn(2+) and Mg(2+) were effective divalent cations while Cl(-) was the most effective anion tested. The enzyme was specific for phosphoglycolate and did not show any activity toward a variety of organic phosphate esters. Growth of the cells on high CO(2) and transfer to air did not result in any significant change in phosphoglycolate phosphatase activity. Competitive inhibition of C. peniocystis triose phosphate isomerase by phosphoglycolate was demonstrated (K(i) = 12.9 micromolar). These results indicate the presence of a specific noninducible phosphoglycolate phosphatase whose sole function may be to hydrolyze phosphoglycolate and prevent phosphoglycolate inhibition of triose phosphate isomerase.