A novel mutant (zea1) of the halotolerant unicellular green alga Dunaliella salina is impaired in the zeaxanthin epoxidation reaction, thereby lacking a number of the β-branch xanthophylls. HPLC analysis revealed that the zea1 mutant lacks neoxanthin (N), violaxanthin (V) and antheraxanthin (A) but constitutively accumulates zeaxanthin (Z). Under low-light physiological growth conditions, the zea1 (6 mg Z per g dry weight or 8 × 10−16 mol Z/cell) had a substantially higher Z content than the wild type (0.2 mg Z per g dry weight or 0.5 × 10−16 mol Z/cell). Lack of N, V, and A did not affect photosynthesis or growth of the zea1 strain. Biochemical analyses suggested that Z constitutively and quantitatively substitutes for N, V, and A in the zea1 strain. This mutant is discussed in terms of its commercial value and potential utilization by the algal biotechnology industry for the production of zeaxanthin, a high-value bioproduct. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 115–124, 2003.