Four multiparous Holstein cows were used in a 4 × 4 Latin square experiment to study the effects of fat sources rich in omega-3 fatty acids on milk production and composition, follicular development, and prostaglandin secretion. All cows were fed a total mixed diet containing 60% grass silage and 40% concentrate. The four treatments were concentrates based either on Megalac, formaldehyde-treated whole linseed, a mixture (50:50, oil basis) of fish oil and formaldehyde-treated whole linseed, or no fat source in the concentrate but with 500 g per day of linseed oil being infused into the duodenum. Feed intakes and milk yield were similar among treatments. In general, the lowest digestibility was observed for the formaldehyde-treated whole linseed treatment. Feeding fish oil decreased milk fat and protein percentages. Alpha-linolenic acid increased from 1.0 to 13.9% of milk fatty acids with linseed oil infusion. This confirms the high potential to incorporate α-linolenic acid into milk, and suggests that the formaldehyde treatment had little effect to limit biohydrogenation in the rumen. Increasing the supply of α-linolenic acid to these cows did not result in an increase in the concentration of eicosapentaenoic acid in milk. Levels of 13,14-dihydro-15-keto-PGF2α in plasma were higher for cows receiving formaldehyde-treated linseed and fish oil. Increases in this metabolite in response to oxytocin challenge, tended to be lower for cows given linseed either as sole oil supplement in the diet or as a duodenal infusion of linseed oil. Follicle dynamics were similar among treatments. Larger corpora lutea (CL) were found with cows that received high levels of omega-3 fatty acids through the diet as formaldehydetreated linseed or as a mixture of formaldehyde-treated linseed and fish oil, although CL were smaller when cows were infused with linseed oil into the duodenum. These results suggest that the improvement in gestation rate that was observed when feeding increased levels of alpha-linolenic acid in earlier work may partly result from lower levels of production of the dienoic prostaglandin PGF2α.