Background: In order to acquire accurate drug pharmacokinetic information, which is required for tissue dosimetry, micro-SPECT must be quantitative to allow for an accurate assessment of radioligand activity in the relevant tissue. This study investigates the feasibility of deriving accurate mouse-specific time-integrated drug pharmacokinetic data in mouse kidneys from activity measurements using micro-SPECT. Methods: An animal experiment was carried out to evaluate the accuracy of 131I activity quantification in mouse kidneys (mean tissue volume of 0.140 mL) using a micro-SPECT system against conventional ex vivo gamma counting (GC) in a NaI(Tl) detector. The imaging setting investigated was that of the mouse biodistribution of a 131I-labelled single-domain antibody fragment (sdAb), currently being investigated for targeted radionuclide therapy of HER2-expressing cancer. SPECT imaging of 131I 365-keV photons was done with a VECTor/CT system (MILabs, Netherlands) using a high-energy mouse collimator with 1.6-mm-diameter pinholes. For both activity quantification techniques, the pharmacokinetic profile of the radioligand from approximately 1 to 73 h p.i. was derived and the time-integrated activity coefficient per gram of tissue (ã/M) was estimated. Additionally, SPECT activity recovery coefficients were determined in a phantom setting. Results: SPECT activities underestimate the reference activities by an amount that is dependent on the 131I activity concentration in the kidney, and thus on the time point of the pharmacokinetic profile. This underestimation is around -12% at 1.5 h (2.89 MBq.mL-1 mean reference activity concentration), -13% at 6.6 h (149 kBq.mL-1), -40% at 24 h (17.6 kBq.mL-1) and -46% at 73 h (5.2 kBq.mL-1) p.i. The ã/M value estimated from SPECT activities is, nevertheless, within -14% from the reference (GC) ã/M value. Furthermore, better quantitative accuracy (within 2% from GC) in the SPECT ã/M value is achieved when SPECT activities are compensated for partial recovery with a phantom-based recovery coefficient of 0.85. Conclusion: The SPECT imaging system used, together with a robust activity quantification methodology, allows an accurate estimation of time-integrated pharmacokinetic information of the 131I-labelled sdAb in mouse kidneys. This opens the possibility to perform mouse-specific kidney-tissue dosimetry based on pharmacokinetic data acquired in vivo on the same mice used in nephrotoxicity studies.