Glucocorticoid treatment had a variety of effects on lipid composition of HeLa S3G (HeLa 65) cells. Total cellular cholesterol levels were lower in treated cultures than in controls within 6 h of the addition of glucocorticoids and remained lower for up to 48 h. Total cell protein and total lipid phosphorus levels were also decreased, but only after more prolonged exposure. The major phospholipid components, viz. phosphatidylcholine and phosphatidylethanolamine, representing, respectively, 48% and 29% of the total phospholipids, were not affected by glucocorticoid treatment. Sphingomyelin, however, increased from 4.8 to 8.3% of the total phospholipid content after 48 h of exposure, whereas lysolecithin increased from 0.1 to 1.9%. The incorporation of labeled precursors was also measured after glucocorticoid addition. As related to cellular protein content, only slight increases were found for all major phospholipids. In contrast, sphingomyelin synthesis increased up to 200% after treatment with glucocorticoids. The fatty acid and plasmalogen compositions were unchanged by glucocorticoids.