Auditory stream segregation refers to the perceptual grouping of sounds, to form coherent representations of objects in the acoustic scene, and is a fundamental aspect of hearing and speech perception. The perceptual segregation of simple interleaved tone sequences has been studied in humans and European starlings (Sturnus vulgaris) using sequences of 2 alternating tones differing in frequency (ABA-ABA-ABA-. . .) The segregation of A and B tones into separate auditory streams is believed to be promoted by preattentive auditory processes that increase the separation of excitation patterns along a tonotopic gradient. We tested the hypothesis that frequency selectivity and forward masking operate as 2 preattentive processes in sequential stream segregation by recording neural responses in the auditory forebrain of awake starlings to repeated ABA- sequences in which we varied the frequency separation (ΔF) between the A and B tones and the tone repetition time (TRT). The A tones were presented at the neurons' characteristic frequency (CF), and B tones differed from the CF over a one-octave range. Larger ΔF values and shorter TRTs promote the perceptual segregation of alternating tone sequences in humans and also resulted in larger differences in neural responses to alternating CF (A) and non-CF (B) tones. Our results are consistent with the hypothesis that preattentive auditory processes, such as frequency selectivity and forward masking, contribute to the perceptual segregation of sequential acoustic events having different frequencies into separate auditory streams, but also suggest that additional processes may be required to account for all known perceptual effects related to sequential auditory stream segregation.