Transient effects in the diffusional quenching of indole fluorescence by acrylamide were detected by frequency-domain fluorometry. The samples are excited with a repetitive train of picosecond pulses, and the frequency response is measured using the harmonic content up to 1 GHz. The decay of unquenched indole in aqueous buffer is dominantly a single exponential. In the presence of acrylamide the partially quenched indole fluorescence became distinctly more complex. Analysis of the frequency response of the fluorescence yields the apparent interaction radius (7.1 Å) and mutual diffusion coefficient (1.1 × 10-5 cm2 s).