A slow fluorescence change of the complex between ras p21 and the fluorescent GTP analogue 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-triphosphate (mGTP) has been postulated to be a signal arising from a step which is rate limiting and precedes the actual GTP hydrolysis reaction [Neal, S. E., Eccleston, J. F., & Webb, M. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565]. We have now shown that the rate of the fluorescence change is accelerated by GTPase-activating protein (GAP) in the same manner as that of the GTP cleavage reaction. In contrast, a faster fluorescence change of smaller amplitude seen in the complex between p21 and the uncleavable 2'(3')-O-(N-methylanthraniloyl)guanosine 5'-O-(beta,gamma-imidotriphosphate) (mGppNHp) is not affected by GAP. The corresponding fluorescent derivative of guanosine 5'-O-(gamma-thiotriphosphate) (mGTP gamma S) shows a very slow fluorescence change after binding to p21, and this rate is also accelerated significantly by GAP. Hydrolysis of GTP gamma S is similarly slow, and it is accelerated by GAP in a similar manner to the fluorescence change. The results are interpreted to indicate that the fluorescence change occurs either at the hydrolysis step or on release of inorganic phosphate or thiophosphate but does not occur in a rate-limiting step preceding hydrolysis.