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Background: Microbrachytherapy enables high local tumor doses sparing surrounding tissues by intratumoral injection of radioactive holmium-166 microspheres (166Ho-MS). Magnetic resonance imaging (MRI) cannot properly detect high local Ho-MS concentrations and single-photon emission computed tomography has insufficient resolution. Computed tomography (CT) is quicker and cheaper with high resolution and previously enabled Ho quantification. We aimed to optimize Ho quantification on CT and to implement corresponding dosimetry. Methods: Two scanners were calibrated for Ho detection using phantoms and multiple settings. Quantification was evaluated in five phantoms and seven canine patients using subtraction and thresholding including influences of the target tissue, injected amounts, acquisition parameters, and quantification volumes. Radiation-absorbed dose estimation was implemented using a three-dimensional 166Ho specific dose point kernel generated with Monte Carlo simulations. Results: CT calibration showed a near-perfect linear relation between radiodensity (HU) and Ho concentrations for all conditions, with differences between scanners. Ho detection during calibration was higher using lower tube voltages, soft-tissue kernels, and without a scanner detection limit. The most accurate Ho recovery in phantoms was 102 ± 11% using a threshold of mean tissue HU + (2 × standard deviation) and in patients 98 ± 31% using a 100 HU threshold. Thresholding allowed better recovery with less variation and dependency on the volume of interest compared to the subtraction of a single HU reference value. Corresponding doses and histograms were successfully generated. Conclusion: CT quantification and dosimetry of 166Ho should be considered for further clinical application with on-site validation using radioactive measurements and intra-operative Ho-MS and dose visualizations. Relevance statement: Image-guided holmium-166 microbrachytherapy currently lacks reliable quantification and dosimetry on CT to ensure treatment safety and efficacy, while it is the only imaging modality capable of quantifying high in vivo holmium concentrations. Key Points: Local injection of 166Ho-MS enables high local tumor doses while sparing surrounding tissue. CT enables imaging-based quantification and radiation-absorbed dose estimation of concentrated Ho in vivo, essential for treatment safety and efficacy. Two different CT scanners and multiple acquisition and reconstruction parameters showed near-perfect linearity between radiodensity and Ho concentration. The most accurate Ho recoveries on CT were 102 ± 11% in five phantoms and 98 ± 31% in seven canine patients using thresholding methods. Dose estimations and volume histograms were successfully implemented for clinical application using a dose point kernel based on Monte Carlo simulations. [ABSTRACT FROM AUTHOR]