This experiment was designed to estimate the apparent transfer of linolenic acid (18:3) from the diet to milk in cows fed fresh and conserved alfalfa forage. Sixteen multiparous Holsteins were involved in a cross over design with three five week periods. Results from the fifth week of each period indicated that cows fed fresh forage had significantly (P < 0.0001) higher levels of 18:3 (1.05 vs. 0.79g/100g fat) and conjugated linoleic acid (CLA)(0.17 vs. 0.07g/100g fat) in their milk compared to cows fed silage. The enrichment of 18:3 in milk from fresh forage fed cows was represented in the lipid concentration of rumen bacteria (6.52% of fatty acids vs. 3.02%). The apparent transfer of 18:3 from diet to milk was 4.36% and 3.81 % for the fresh and silage groups respectively. Fresh forage feeding resulted in milk with lower saturated, and higher polyunsaturated fatty acids, particularly CLA and 18:3, compared to similar forage that had been ensiled.