Inflammation worsens joint destruction in osteoarthritis (OA) and aggravates pain. Although n-3 fatty acids reduce inflammation, different n-3 fatty acids have different effects on inflammation and clinical outcomes, with eicosapentaenoic acid (EPA) having the strongest effect. We examined whether specific essential fatty acid levels affected the development of OA. We studied participants from the Multicenter Osteoarthritis Study (MOST) at risk of developing knee OA. As part of MOST, participants were asked repeatedly about knee pain, and knee radiographs and magnetic resonance images (MRIs) were obtained. Using baseline fasting samples, we analyzed serum fatty acids with standard assays. After excluding participants with baseline OA, we defined two sets of cases based on their status through 60 months' follow-up: those developing incident radiographic OA and those developing incident symptomatic OA (knee pain and radiographic OA). Controls did not develop these outcomes. Additionally, we examined worsening of MRI cartilage damage and synovitis and worsening knee pain and evaluated the number of hand joints affected by nodules. In regression models, we tested the association of each OA outcome with levels of specific n-3 and n-6 fatty acids, adjusting for age, sex, body mass index, education, physical activity, race, baseline pain, smoking, statin use, and depressive symptoms. We studied 363 cases with incident symptomatic knee OA and 295 with incident radiographic knee OA. The mean age was 62 years (59% women). We found no associations of specific n-3 fatty acid levels, including EPA, or of n-6 fatty acid levels with incident OA (eg, for incident symptomatic knee OA, the odds ratio per SD increase in EPA was 1.0 [95% confidence interval 0.87-1.17]). Results for other OA outcomes also failed to suggest a protective effect of specific n-3 fatty acids with OA outcomes. We found no association of serum levels of EPA or of other specific n-3 fatty acids or n-6 fatty acids with risk of incident knee OA or other OA outcomes.