Milk fat is high in saturated fatty acids (SFA) and high intakes of SFA are associated with cardiovascular diseases. The aim of the present study was to prospectively evaluate the potential risk of a first-ever acute myocardial infarction (AMI) in relation to the estimated milk-fat intake, reflected as the proportions of pentadecanoic acid (15:0) and heptadecanoic acid (17:0) in serum lipid esters. This was evaluated in a study population selected within the Västerbotten Intervention Program and the northern Sweden ‘Monitoring of Trends and Determinants in Cardiovascular disease’ survey populations. A prospective case–control design was used. The proportions of the biomarkers were lower in the cases (n78) than in the controls (n156), who were matched for age, sex, sampling time and geographical region. The standardised odds ratios of becoming an AMI case were between 0·7 and 0·8 for the biomarkers. The proportions of 15:0 and 17:0 in serum phospholipids were significantly and negatively correlated to serum concentrations of plasminogen activator inhibitor-1, tissue-type plasminogen activator, triacylglycerols, insulin, specific insulin, pro-insulin and leptin (all P<0·0001), suggesting a negative relationship to the insulin-resistance syndrome and the risk of CHD. Adjustment for BMI did not materially change the relationships. Although there seems to be a negative association between milk-fat intake as mirrored by the proportions of 15:0 and 17:0 in serum lipid esters and a first-ever AMI, adjustment for clinical risk factors removed this relationship.