In examining substances causing hemoglobin (Hb) denaturation in vitro obtained from lipophilic fractions of fishes, the transformation of oxyhemoglobin (O₂Hb) to methemoglobin (MetHb) was observed in the ether extracts of Engraulis japonica (anchovy) and the viscera of Cololabis saira in six fish species. Removal of acidic substances from ether extracts of anchovy viscera by pretreatment with Na₂CO₃ resulted in suppression of Hb denaturation activity, suggesting that the substance causing Hb denaturation was acidic. The transformation of hemoglobin was observed in three fractions separated by thin layer chromatography : Fr. 1, found at the origin contained primarily phospholipids ; Fr. 2 consisted of two bands between fractions of phospholipids and free fatty acids ; and Fr. 3 was composed of free fatty acids. Addition of α-tocopherol to the sample completely suppressed the transformation of O₂Hb to MetHb, and the usual brownish precipitation caused by protein denaturation did not appear. The concentrations of free eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) contained in ether extracts were directly proportional to magnitude of the MetHb formation index. These results indicated that the main acidic substances causing blood Hb denaturation in vitro were polyunsaturated fatty acids such as EPA and DHA which were present in abundance in anchovy viscera.