Enrichment of milk fat with n-3 fatty acids, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), may be advantageous because of their beneficial effects on human health. In addition, these fatty acids play an important role in reproductive processes in dairy cows. Our objective was to evaluate the protection of EPA and DHA against rumen biohydrogenation provided by Ca salts of fish oil. Four Holstein cows were assigned in a Latin square design to the following treatments: 1) ruminal infusion of Ca salts of fish oil and palm fatty acid distillate low dose (CaFO-1), 2) ruminal infusion of Ca salts of fish oil and palm fatty acid distillate high dose (CaFO-2), 3) ruminal infusion of fish oil high dose (RFO), and 4) abomasal infusion of fish oil high dose (AFO). The high dose of fish oil provided approximately 16 and approximately 21 g/d of EPA and DHA, respectively, whereas the low dose (CaFO-1) provided 50% of these amounts. A 10-d pretreatment period was used as a baseline, followed by 9-d treatment periods with interceding intervals of 10 d. Supplements were infused every 6 h, milk samples were taken the last 3 d, and plasma samples were collected the last day of baseline and treatment periods. Milk fat content of EPA and DHA were 5 to 6 times greater with AFO, but did not differ among other treatments. Milk and milk protein yield were unaffected by treatment, but milk fat yield and DM intake were reduced by 20 and 15%, respectively, by RFO. Overall, results indicate rumen biohydrogenation of long chain n-3 fatty acids was extensive, averaging >85% for EPA and >75% for DHA for the Ca salts and unprotected fish oil supplements. Thus, Ca salts of fish oil offered no protection against the biohydrogenation of EPA and DHA beyond that observed with unprotected fish oil; however, the Ca salts did provide rumen inertness by preventing the negative effects on DM intake and milk fat yield observed with unprotected fish oil.