The rotational dynamics of a phenytoin fluoroimmunoassay system is investigated by using multifrequency phase and modulation fluorescence spectroscopy. Results indicate that the fluorescein label on this relatively small hapten undergoes significant (>45%) local motion even when bound to the phenytoin antibody. We attribute this local motion to unhindered rotation of the fluorescein moiety around the bond attaching it to phenytoin. This local motion affects the sensitivity of a steady-state polarization immunoassay dramatically.