The kinetics and efficiency of sterol production and bioconversion of phytosterols in two heterotrophic protists Oxyrrhis marina and Gyrodinium dominans were examined by feeding them two different algal species (Rhodomonas salina and Dunaliella tertiolecta) differing in sterol profiles. R. salina contains predominantly brassicasterol (≌99%) and <2% cholesterol. The major sterols in D. tertiolecta are ergosterol (45–49%), 7-dehydroporiferasterol (29–31%) and fungisterol (21–26%). O. marina fed R. salina metabolized dietary brassicasterol to produce 22-dehydrocholesterol and cholesterol. O. marina fed D. tertiolecta metabolized dietary sterols to produce cholesterol, 22-dehydrocholesterol, brassicasterol and stigmasterol. G. dominans fed either R. salina or D. tertiolecta metabolized dietary sterols to make cholesterol, brassicasterol and a series of unknown sterols. When protists were fed R. salina, which contains cholesterol, the levels of cholesterol were increased to a magnitude of nearly 5- to 30-fold at the phytoplankton-heterotrophic protist interface, equivalent to a production of 172.5 ± 16.2 and 987.7 ± 377.7 ng cholesterol per mg R. salina carbon consumed by O. marina and G. dominans, respectively. When protists were fed D. tertiolecta, which contains no cholesterol, a net production of cholesterol by the protists ranged from 123.2 ± 30.6 to 871.8 ± 130.8 ng per mg algal C consumed. Cholesterol is not only the dominant sterol, but a critical precursor for many physiologically functional biochemicals in higher animal. As intermediates, these heterotrophic protists increase the amount of cholesterol at the phytoplankton-zooplankton interface available to higher trophic levels relative to zooplankton feeding on algae directly.