During hunting, insectivorous bats such as Eptesicus fuscus progressively increase the pulse repetition rate, shorten the pulse duration, and lower the frequency and amplitude of emitted pulses as they search, approach and finally intercept insects or negotiate obstacles. As such, analysis of an echo parameter by the bat is inevitably affected by other co-varying echo parameters. The present study examined the effect of pulse duration on frequency selectivity of neurons in the central nucleus of the inferior colliculus (IC) of the big brown bat. A family of iso-level frequency tuning curves of each IC neuron was first measured with tone bursts of different durations. The bandwidth of iso-level frequency tuning curves within each family was then compared. Our data show that most IC neurons discharge maximally to a particular pulse duration which is defined as the best duration (BDu). The iso-level frequency tuning curves of these duration-selective neurons have the narrowest bandwidth when measured with the BDu pulse than with non-BDu pulses. They also have the narrowest bandwidth when measured with a short than with a long BDu pulse. These data suggest that frequency selectivity of duration-selective IC neurons becomes sharper when short echo duration at the final phase of hunting is encoded by IC neurons that have short BDu.