We present new Rossi X-ray Timing Explorer observations of the low-mass X-ray binary 4U 1608–52 during the decay of its 1998 outburst . We detect by a direct FFT method the existence of a second kilohertz quasi-periodic oscillation ( kHz QPO ) in its power density spectrum , previously only seen by means of the sensitivity-enhancing ‘ shift and add ’ technique . This result confirms that 4U 1608–52 is a twin kHz QPO source . The frequency separation between these two QPO decreased significantly , from 325.5 \pm 3.4 Hz to 225.3 \pm 12.0 Hz , as the frequency of the lower kHz QPO increased from 470 Hz to 865 Hz , in contradiction with a simple beat-frequency interpretation . This change in the peak separation of the kHz QPOs is closely similar to that previously seen in Sco X–1 , but takes place at a ten times lower average luminosity . We discuss this result within the framework of models that have been proposed for kHz QPO . Beat frequency models where the peak separation is identified with the neutron star spin rate , as well as the explanations previously proposed to account for the similar behavior of the QPOs in Sco X–1 , are strongly challenged by this result .