We measured total cholesterol levels in serum and disodium ethylenediaminetetraacetate (EDTA) plasma samples obtained from 84 healthy medical students during their entrance physical examinations and 48 adults from a cholesterol screening program who were resampled because they had initial values of 5.17 mmol/L (200 mg/dL) or higher. Plasma cholesterol concentrations were 4.7% lower than those in serum samples. The plasma and serum values were highly correlated (r=.994), however, suggesting that measurements in EDTA plasma can be converted readily to equivalent serum concentrations. Thus, the negative bias in EDTA plasma was greater than the 3% value cited in the National Cholesterol Education Program Guidelines, probably because the amount of EDTA now provided in evacuated blood collection tubes is 50% greater than in those used when the 3% value was established. These findings are relevant to the interpretation of both cholesterol screening measurements and follow-up lipoprotein analyses.