Cell-free preparations of Tetrahymena thermophila catalyze the direct desaturation of cholesterol to Delta(7)-dehydrocholesterol (provitamin D-3) The activity was isolated in the microsomal fraction from Tetrahymena homogenates. Delta(7)-Desaturase activity was stimulated fivefold by the addition of 6 mM ATP. Other cofactors assayed, including NAD, NADP, NADH or NADPH, had no significant effect. The activity was found in microsomes prepared from stationary-phase cultures of the ciliate? grown either with or without added cholesterol, thus indicating that it is constitutively expressed in T. themophila cells.