The effects of a wide variety of oxygenated sterols upon sterol biosynthesis and hydroxymethylglutaryl-CoA reductase (mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34) activity in a wild-type clone and in a 25-hydroxycholesterol-resistant clone of Chinese hamster lung (Dede) cells are described. Derivatives of cholesterol which were oxygenated in the 6, 7 or 15 positions of the sterol nucleus or in the 20, 22, 24 or 25 positions of the sterol side chain were shown to be potent inhibitors of sterol synthesis and reductase activity in the wild-type cells but none of these substitutions had any effect on the 25-hydroxycholesterol-resistant A2 clone. A 32-hydroxylated derivative of lanosterol also suppressed sterol synthesis and reductase activity in wild-type cells but had no significant effect upon the A2 line. It was also appraent that a complete sterol side chain was necessary for inhibitory activity. Studies of a wide range of inhibitory sterols indicated that there was a close correlation between their effects upon sterol synthesis and reductase activity and that their inhibitory action was specific for sterol biosynthesis since little effect was observed upon fatty acid or CO2 synthesis. Previous studies had shown that the uptake of 25-hydroxycholesterol by the resistant A2 line was unimpaired and the present results indicate that metabolism of this oxygenated sterol is also unaltered. These results, in conjunction with previous studies, suggest that the resistant A2 line is defective in feedback regulation of cholesterol synthesis and that all of the oxygenated sterols tested suppress the biosynthetic pathway through at least one common step.