Loop L5 of kinesin is located near the ATPase site, in common with kinesins of various animal species. The rice plant-specific kinesin K16 also has a corresponding loop that is slightly shorter than that of mouse brain kinesin. The present study was designed to monitor conformational changes in loop L5 during ATP hydrolysis. For this purpose, we introduced one reactive cysteine into the L5 of rice kinesin and modified it with fluorescent probes. The cysteine in L5 was labeled with a fluorescent probe 2-(4′(iodoacetamide) anilino-naphthalene-6-sulufonic acid sodium salt) [IAANS]. IAANS was incorporated into L5 at an almost equimolar ratio in the absence of nucleotides. In contrast, the incorporated amount was reduced to 0.62 and 0.32 mol IAANS/mol motor domain in the presence of ATP and ADP, respectively. Upon nucleotide addition, the fluorescent intensity of IAANS incorporated into L5 was significantly reduced to 63% and 51% for ATP and ADP, respectively. These results suggest that L5 of rice kinesin significantly changes its conformation during ATP hydrolysis.