Dietary polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which have been attributed to their availability to modulate Na⁺, Ca²⁺, and several K⁺ channels. However, their effects on HERG channels are unknown. In this study we have analyzed the effects of arachidonic (AA, -6) and docosahexaenoic acids (DHA, -3) on HERG channels stably expressed in CHO cells by using the whole-cell patch-clamp technique. At 10 µM, AA and DHA blocked HERG channels, at the end of 5-s pulses to -10 mV, to a similar extent (37.7±2.4% vs 41.4±9.3%, n=7-10, p>0.05). ETYA, a nonmetabolizable AA analog, induced similar effects than AA on HERG current. Both PUFAs shifted the midpoint of activation curves of HERG channels by -5.1±1.8 mV (n=10, p<0.05) and -11.2±1.2 mV (n=7, p<0.01). Also, AA and DHA shifted the midpoint of inactivation curves by +12.0±3.9 mV (n=4; p<0.05) and +15.8±4.3 mV (n=4; p<0.05), respectively. DHA and AA accelerated the deactivation kinetics and slowed the inactivation kinetics at potentials positive to +40 mV. Block induced by DHA, but not that produced by AA, was higher when measured after applying a pulse to -120 mV (IO). Finally, both AA and DHA induced a use-dependent inhibition of HERG channels. In summary, block induced by AA and DHA was time-, voltage- and use-dependent. The results obtained suggest that both PUFAs bind preferentially to the open state of the channel, although an interaction with inactivated HERG channels cannot be ruled out for AA.