The aim of this study was to evaluate the potential of anatomy-based reconstruction, using microCT information, to improve quantitative accuracy in multiple-pinhole SPECT. Multiple-pinhole SPECT and microCT images were acquired with the Micro Deluxe Phantom using both hot and cold rod inserts. The phantoms were filled with 3.7 MBq/ml of (99m)Tc. To improve microCT contrast, the phantoms were also filled with contrast agent. Emission images were reconstructed using a one-step-late (OSL) modification of the ordered subsets expectation maximization (OSEM) algorithm for incorporation of microCT information, to encourage smoothing within but not across boundaries. To allow quantification, the OSL OSEM algorithm takes into account imperfect camera motion, collimator response, angular variation of the sensitivity, intrinsic camera resolution, attenuation and scatter. For comparison, the emission images were also reconstructed by OSEM using post-reconstruction filtering and by OSL OSEM using a quadratic prior and an edge-preserving prior. In each rod of the phantoms the recovery coefficient (RC), defined as measured divided by the true activity concentration, was expressed as a function of the noise. Different noise levels were obtained by varying the amount of spatial filtering during or after reconstruction and by the use of binominal deviates. Compared to conventional OSEM using post-reconstruction filtering and compared to OSL OSEM using a quadratic prior, our study demonstrated that the use of anatomical information during reconstruction significantly improved the quantitative accuracy in both cold and hot rods with a diameter larger than or equal to 2.4 mm. When compared to the edge-preserving prior, the anatomical prior performs significantly better for hot rods with a diameter a parts per thousand yen2.4 mm. For the 4.0-mm hot rods for example, the RC averaged over the different noise levels was 0.67 +/- 0.02 when multiple-pinhole SPECT images were reconstructed using anatomical information, compared to 0.54 +/- 0.08, 0.60 +/- 0.04 and 0.64 +/- 0.02 when OSEM in combination with a post-reconstruction filter, OSL OSEM using a quadratic prior and OSL OSEM using a median root prior was used, respectively. For the 4.0-mm cold rods, the RC averaged over the different noise levels was 0.61 +/- 0.03 when the multiple-pinhole SPECT images were reconstructed using anatomical information, compared to 0.54 +/- 0.07, 0.53 +/- 0.08 and 0.60 +/- 0.03 when OSEM in combination with a post-reconstruction filter, OSL OSEM using a quadratic prior and OSL OSEM using a median root prior was used, respectively. Anatomy-based reconstruction using microCT information has the potential to improve quantitative accuracy in multiple-pinhole SPECT.