Background: Quantitative activity estimation is essential in targeted radionuclide therapy dosimetry. Misregistration between SPECT and CT images at the same imaging time point due to patient movement degrades accuracy. This work aims to study the mismatch effects between CT and SPECT data on attenuation correction (AC), volume-of-interest (VOI) delineation and registration for activity estimation.Methods: Nine 4D XCAT phantoms were generated at 1, 24, and 144 hrs post In-111 Zevalin injection, varying in activity distributions, body and organ sizes. Realistic noisy SPECT projections were generated by an analytical projection and reconstructed with quantitative OS-EM method. CT images were shifted from -5 to 5 voxels as well as according to clinical reference corresponding to SPECT images at each time point. For AC effect, mismatched CT images were used for AC in SPECT reconstruction while VOIs were mapped out from matched CTs. For VOI effect, target organs were mapped out using mismatched CTs with matched CTs for AC. For registration effect, non-rigid registrations were performed on sequential mismatched CTs to align corresponding SPECT images, with no AC and VOI mismatch. Bi-exponential curve fitting was performed to obtain time-integrated activity (TIA). Organ activity errors (%OAE) and TIA errors (%TIAE) were calculated.Results: According to clinical reference, %OAE was larger for organs near ribs for AC effect, e.g., -2.58%±0.81% for liver. For VOI effect, %OAE was larger for small and low uptake organs, e.g., -11.94%±10.34% for spleen. %OAE was proportional to mismatch magnitude, e.g., 4.77%±1.41%, 12.01%±3.97% and 42.81%±6.38% for 1-, 2-, and 5-voxel mismatch for lungs. For registration effect, %TIAE were larger when mismatch existed in more numbers of SPECT/CT images, while no substantial difference was observed when using mismatched CT at different time points for registration reference. %TIAE was highest for VOI, followed by registration and AC, e.g., 37.61%±5.08%, 14.25%±7.07% and 1.13%±0.90% respectively for kidneys.Conclusions: The mismatch between CT and SPECT images poses a significant impact on accuracy of quantitative activity estimation in dosimetry, attributed particularly from VOI delineation errors. It is recommended to perform registration between emission and transmission images at the same time point to ensure dosimetric accuracy.