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STUDY DESIGN.: In vitro biomechanical study. OBJECTIVE.: To characterize cervical total disc replacement (TDR) kinematics above two-level fusion, and to determine the effect of fusion alignment on TDR response. SUMMARY OF BACKGROUND DATA.: Cervical TDR may be a promising alternative for a symptomatic adjacent level after prior multilevel cervical fusion. However, little is known about the TDR kinematics in this setting. METHODS.: Eight human cadaveric cervical spines (C2-T1, age: 59 ± 8.6 years) were tested intact, after simulated two-level fusion (C4-C6) in lordotic alignment and then in straight alignment, and after C3-C4 TDR above the C4-C6 fusion in lordotic and straight alignments. Fusion was simulated using an external fixator apparatus, allowing easy adjustment of C4-C6 fusion alignment, and restoration to intact state upon disassembly. Specimens were tested in flexion-extension using hybrid testing protocols. RESULTS.: The external fixator device significantly reduced range of motion (ROM) at C4-C6 to 2.0 ± 0.6°, a reduction of 89 ± 3.0% (P < 0.05). Removal of the fusion construct restored the motion response of the spinal segments to their intact state. The C3-C4 TDR resulted in less motion as compared to the intact segment when the disc prosthesis was implanted either as a stand-alone procedure or above a two-level fusion. The decrease in motion of C3-C4 TDR was significant for both lordotic and straight fusions across C4-C6 (P < 0.05). Flexion and extension moments needed to bring the cervical spine to similar C2 motion endpoints significantly increased for the TDR above a two-level fusion compared to TDR alone (P < 0.05). Lordotic fusion required significantly greater flexion moment, whereas straight fusion required significantly greater extension moment (P < 0.05). CONCLUSION.: TDR placed adjacent to a two-level fusion is subjected to a more challenging biomechanical environment as compared to a stand-alone TDR. An artificial disc used in such a clinical scenario should be able to accommodate the increased moment loads without causing impingement of its endplates or undue wear during the expected life of the prosthesis. [ABSTRACT FROM AUTHOR]