The authors have previously reported that the L4–5 isthmic spondylolisthesis lesion often progresses more than the L5-S1 lesion in adult patients. This biomechanical study compares the in vitro stability of the L4–5 isthmic spondylolisthesis lesion compared with the L5-S1 isthmic lesion. The authors also analyzed the role of the L5 iliolumbar ligament as a contributing factor to stability. Six fresh frozen human cadaveric specimens (L4 to the sacrum including the iliolumbar ligamentous complex) were tested by applying 10 Nm flexion-extension moments. Sagittal plane motion was measured with the specimens intact and after sequential transection of the pars interarticulares at L4 and L5 and finally with the iliolumbar ligaments cut at L5-S1. L4–5 and L5-S1 both showed significant increases in rotation with the pars defect compared with normal (L4–5 = +2.0, L5-S1 = +3.2 degrees). Decreased translation of L5-S1 occurred with pars defect at this level. There were no significant differences at the L5-S1 level after sectioning of the iliolumbar ligament. Calculating the percentage difference from normal, L4–5 with a pars defect exhibited significantly greater relative motion compared with L5-S1 with the same defect; 12% more rotation, 33% more shear, and 43% more axial translation. The iliolumbar ligament did not appear to contribute to these diferences because there was no significant change in the L5-S1 kinematics after its transection. These results support the hypothesis that L4–5 pars defects are more unstable than L5-S1 lesions. The ilioumbar ligament could not be implicated as the major contributing factor in these differences.