PUFA have been associated with lower risk of CVD, but less is known about their association with stroke risk. Fish, a major source of n-3 PUFA, may also contain methylmercury, which has been associated with higher risk of CVD and attenuation of the benefits of long-chain n-3 PUFA. We investigated the associations of serum n-3 and n-6 PUFA and hair Hg with risk of stroke in men. A total of 1828 men from the prospective, population-based Kuopio Ischaemic Heart Disease Risk Factor Study, aged 42–60 years and free of CVD at baseline in 1984–1989 were studied. Cox regression models were used for the analyses. During the mean follow-up of 21·2 years, 202 stroke cases occurred, of which 153 were ischaemic strokes. After adjustment for age and examination year, the only statistically significant association among the n-3 and n-6 PUFA was observed between the n-3 PUFA α-linolenic acid and risk of haemorrhagic stroke (hazard ratio in the highest v. the lowest quartile 0·33; 95 % CI 0·13, 0·86; Ptrend=0·03). However, further adjustments attenuated the association to statistically non-significant. Hair Hg was not associated with stroke risk, but among those with hair Hg above the median level, higher serum long-chain n-3 PUFA concentrations were associated with a higher risk of ischaemic stroke. In our cohort of men, serum n-3 or n-6 PUFA or hair Hg were not associated with stroke risk; however, the interaction between Hg and long-chain n-3 PUFA with regard to ischaemic stroke risk warrants further investigation.