Purpose SPECT/CT is a hybrid imaging modality, which combines a 3D scintigraphy (SPECT) and a conventional computerised tomography (CT). SPECT/CT allows accurate anatomical localisation of metabolic tracer activity. It allows the correlation of surgical factors such as tunnel position and orientation with mechanical alignment, clinical outcome and biological factors. The purpose of this study was to investigate whether the SPECT/CT tracer uptake (intensity and distribution) correlates with the stability and laxity of the knee joint and the position and orientation of the tibial and femoral tunnels in patients after anterior cruciate ligament (ACL) reconstruction. Methods A consecutive series of knees (n066), with symptoms of pain and/or instability after ACL reconstruction were prospectively evaluated using clinical examination and 99mTc-HDP-SPECT/CT. Clinical laxity testing was performed using the Rolimeter (Ormed, Freiburg, Germany) including Lachman testing (0-2 mm, 3-5 mm, 6-10 mm, >10 mm), anterior drawer test (0-2 mm, 3-5 mm, 6-10 mm, >10 mm), pivot shift test (positive versus negative) and patientbased subjective instability (yes versus no).For analysis of SPECT/CT tracer uptake a previously validated SPECT/CT localisation scheme consisting of 17 tibial, nine femoral and four patellar regions on standardised axial, coronal, and sagittal slices was used. The tracer activity on SPECT/CT was localised and recorded using a 3D volumetric and quantitative analysis software.Mean, standard deviation, minimum and maximum of grading for each area of the localisation scheme were recorded. The position and orientation of the tibial and femoral tunnel was assessed using a previously published method on 3D-CT. Results Correlation of instability, pivot shift as well as clinical laxity testing with 99mTc-HDP-SPECT/CT tracer uptake intensity and distribution showed no significant correlation. 99mTc-HDP-SPECT/CT tracer uptake correlated significantly with the position and orientation of the ACL graft. A more horizontal femoral graft position showed significantly increased tracer uptake within the superior and posterior femoral regions. A more posteriorly-placed femoral insertion site showed significantly more tracer uptake within the femoral and tibial tunnel regions. A more vertical or a less medial tibial tunnel orientation showed significant increased uptake within the tibial and femoral tunnel regions. A more anterior tibial tunnel position showed significantly more tracer uptake in the femoral and tibial tunnel regions as well as the entire tibiofemoral joint. Conclusions SPECT/CT tracer uptake intensity and distribution showed a significant correlation with the femoral and tibial tunnel position and orientation in patients with symptomatic knees after ACL reconstruction. No correlation was found with stability or clinical laxity.Michael T. Hirschmann and Dominic Mathis contributed equally to this paper.