The stratification of layers of differing prominence (foreground/background) is a common technique in orchestration. Musicians heard 23 excerpts containing foreground and background layers as previously determined by music analysts. A given layer comprised either a single auditory stream of one or more blended instruments or a harmonic or rhythmic background. Two-layer excerpts had either the same, overlapping, or different instrument families (timbre class). First, musicians rated the perceived degree of segregation of musical materials in two-layer and single-stream excerpts. Second, they heard each of the two layers in isolation and then together and rated the relative prominence of the layers. Heterogeneous instrument combinations yielded the greatest difference in relative prominence, followed by overlapping and then homogeneous combinations. Acoustic and score-based descriptors were extracted to quantify their relative contribution to perceptual stratification. Timbre class and between-layer differences in timbre and dynamics played a role, providing evidence of how timbral differences enhance relative prominence in orchestral music. Perceptual segregation was positively but very weakly related to relative prominence, supporting findings that although segregation is necessary to form layers, this mechanism is separable from that which places the streams into the same representational space to allow for the assessment of their relative prominence.