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To expand the availability of SPECT for biomedical research, we developed a SPECT imaging system on an existing animal PET detector by adding a slit-slat collimator. As the detector crystals are pixelated, the relative slat-to-crystal position (SCP) in the axial direction affects the photon flux distribution onto the crystals. The accurate knowledge of SCP is important to the axial resolution and sensitivity of the system. This work presents a method for optimizing SCP in system design and for determining SCP in system geometrical calibration. The optimization was achieved by finding the SCP that provides higher spatial resolution in terms of average-root-mean-square (R̅M̅S̅) width of the axial point spread function (PSF) without loss of sensitivity. The calibration was based on the least-square-error method that minimizes the difference between the measured and modeled axial point spread projections. The uniqueness and accuracy of the calibration results were validated through a singular value decomposition (SVD) based approach. Both the optimization and calibration techniques were evaluated with Monte Carlo (MC) simulated data. We showed that the [R̅M̅S̅] was improved about 15% with the optimal SCP as compared to the least-optimal SCP, and system sensitivity was not affected by SCP. The SCP error achieved by the proposed calibration method was less than 0.04 mm. The calibrated SCP value was used in MC simulation to generate the system matrix which was used for image reconstruction. The images of simulated phantoms showed the expected resolution performance and were artifact free. We conclude that the proposed optimization and calibration method is effective for the slit-slat collimator based SPECT systems.