The accumulation of different precursors of carotenoid biosynthesis in carotenoid-deficient mutants of Chlamydomonas reinhardtii was studied by HPLC-analysis. ζ-Carotene accumulated in several ac5 mutants, this character cosegregated with mutations in the ac5 gene. Two groups of ac5 mutants differing in ζ-carotene accumulation were distinguished. One (ac5-1) accumulated ζ-carotene in the dark but not in the light. The other (ac5-2) accumulated ζ-carotene under both dark and light conditions. ac5-2 strains accumulated more ζ-carotene in the dark than ac5-1 strains. Genetic data suggested that the mutations ac5-1 and ac5-2 were allelic. Pleiotropic effects of mutations in the ac5 gene included decreased levels of chlorophyll a and b and acetate requirement. The results are consistent with the presence of a defective ζ-carotene desaturase in ac5 mutants.