Background: Data on the relation of plasma and dietary omega-3 (n-3) fatty acids (FAs) with heart failure (HF) risk have been inconsistent. Objective: We evaluated the relation of n23 FAs with HF in US male physicians. Design: We used nested case-control (n = 1572) and prospective cohort study designs (n = 19,097). Plasma phospholipid n-3 FAs were measured by using gas chromatography, and food-frequency questionnaires were used to assess dietary n-3 FAs and fish intake. Incident HF was ascertained via annual follow-up questionnaires and validated in a subsample. Results: The mean age was 58.7 y at blood collection. In a multivariable model, plasma α-linolenic acid (ALA) was associated with a lower risk of HF in a nonlinear fashion (P-quadratic trend = 0.02), and the lowest OR was observed in quintile 4 (0.66; 95% CI: 0.47, 0.94). Plasma EPA and DHA were not associated with HF, whereas plasma docosapentaenoic acid (DPA) showed a nonlinear inverse relation with HF for quintile 2 (OR: 0.55; 95% CI: 0.39, 0.79). Dietary marine n-3 FAs showed a trend toward a lower risk of HF in quintile 4 (HR: 0.81; 95% CI: 0.64, 1.02) and a nonlinear pattern across quintiles. Fish intake was associated with a lower risk of HF, with RRs of ∼0.70 for all categories of fish consumption greater than one serving per month. Conclusions: Our data are consistent with an inverse and nonlinear relation of plasma phospholipid ALA and DPA, but not EPA or DHA, with HF risk. Fish consumption greater than once per month was associated with a lower HF risk.