We describe a simple, direct, and specific manual method for quantitative determination of total cholesterol in serum. In the method, which requires no extraction of the serum, a single stable reagent and 50 µl of sample is used. Serum cholesterol concentrations determined by this method did not differ statistically from those obtained by the reference method of Abell et al. Hemoglobin, bilirubin, and γ-globulin do not interfere unless present at markedly supranormal concentrations. Inter-run precision is about ±3% (95% confidence limits).