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Studies were conducted on the underlying mechanism for differences in serum cholesterol in two lines of chickens. The serum level of free and combined cholesterol was higher in the high line than the low line; no significant differences were observed in the serum level of fatty acids or phospholipids. The lines did not differ in the weights of several organs or in the cholesterol level of the adrenal, liver, brain, or testis. The high line had a higher serum cholesterol level than the low line, from hatching to 16 weeks of age. The lines did not differ in their response to fasting, which makes it improbable that a dietary influence accounts for the line differences. They responded similarly to injections of testosterone, estrogens, cortisone, or thyroxin.