Estrogen replacement therapy (ERT), which produces acquired resistance to activated protein C when superimposed on heritable resistance to activated protein C (the mutant Factor V Leiden trait), may promote venous and arterial thrombosis. In a cross-sectional study of 423 women referred for hyperlipidemic therapy (93 of whom [22%] were on ERT), our specific aim was to determine whether ERT and heterozygosity for the Factor V Leiden mutation and/or resistance to activated protein C interacted as risk factors for atherothrombosis. Of the 423 women, 168 (40%) had atherothrombosis, 19 (4%) were heterozygous for Factor V Leiden mutation or had resistance to activated protein C <2 (Factor V Leiden mutation+), and 404 were wild-type normal for the Factor V gene and/or had resistance to activated protein C > or =2 (Factor V Leiden mutation-). By stepwise logistic regression, positive explanatory variables for atherothrombosis included hypertension (p = 0.002), age (p = 0.003), relatives with atherothrombosis (p = 0.002), anticardiolipin antibody immunoglobulin-M (p = 0.02), and a Factor V Leiden mutation*ERT interaction term where atherothrombosis events were more likely in 2 subgroups of women (ERT- and Factor V Leiden mutation-) or (ERT+ and Factor V Leiden mutation+) (p = 0.02). High-density lipoprotein cholesterol was inversely associated with atherothrombosis (p = 0.004). In a separate logistic regression model for the 213 women with a polymerase chain reaction measurement of the Factor V gene, ERT was protective (p = 0.008); the Factor V Leiden mutation was positively associated with atherothrombosis (p = 0.05). The atherothrombosis odds ratio risk for ERT (yes vs no) was 0.36 (95% confidence intervals [CI] 0.16 to 0.74, p = 0.007). The atherothrombosis risk odds ratio in women heterozygous for the Factor V Leiden mutation (vs normal) was 2.00 (95% CI 1.02 to 4.22, p = 0.05). ERT may be protective against atherothrombosis when the Factor V Leiden mutation is absent, whereas the Factor V Leiden mutation may increase risk for atherothrombosis, particularly in the presence of ERT. We suggest that the Factor V Leiden mutation be measured in all women on ERT or before beginning ERT to identify those heterozygous for the Factor V Leiden mutation (4%), in whom ERT is relatively or absolutely contraindicated because of increased risk for atherothrombosis and thromboembolism. A second, much larger group of women will also be identified without the factor V Leiden mutation (96%), in whom ERT may reduce the risk for atherothrombosis.