Recently, conformational changes of the amino-terminal helix (A'alpha helix), in addition to the reported conformational changes of the carboxyl-terminal helix J alpha helix), have been proposed to be important for the regulatory function of the light-oxygen-voltage 2 domain (LOV2) of phototropin 1 from Arabidopsis. However, the reaction dynamics of the A'alpha helix have not been examined. Here, the unfolding reactions of the A'alpha and J alpha helices of the LOV2 domain of phototropin 1 from Arabidopsis thaliana were investigated by the time-resolved transient grating (TG) method. A mutant (T469I mutant) that renders the A'alpha helix unfolded in the dark state showed unfolding of the J alpha helix with a time constant of 1 ms, which is very similar to the time constant reported for the wildtype LOV2-linker sample. Furthermore, a mutant (I608E mutant) that renders the J alpha helix unfolded in the dark state exhibited an unfolding process of the A'alpha helix with a time constant of 12 ms. On the basis of these experimental results, it is suggested that the unfolding reactions of these helices occurs independently.