Abstract An important aspect of auditory scene analysis is the sequential grouping of similar sounds into one “auditory stream” while keeping competing streams separate. In the present low-noise fMRI study we presented sequences of alternating high-pitch (A) and low-pitch (B) complex harmonic tones using acoustic parameters that allow the perception of either two separate streams or one alternating stream. However, the subjects were instructed to actively and continuously segregate the A from the B stream. This was controlled by the additional instruction to listen for rare level deviants only in the low-pitch stream. Compared to the control condition in which only one non-separable stream was presented the active segregation of the A from the B stream led to a selective increase of activation in the left auditory cortex (AC). Together with a similar finding from a previous study using a different acoustic cue for streaming, namely timbre, this suggests that the left auditory cortex plays a dominant role in active sequential stream segregation. However, we found cue differences within the left AC: Whereas in the posterior areas, including the planum temporale, activation increased for both acoustic cues, the anterior areas, including Heschl’s gyrus, are only involved in stream segregation based on pitch.