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A method using line sources to calibrate a pinhole MicroSPECT system was proposed. Projections' coordinates of "vitual" point sources were obtained by solving equations of centerlines, which were detected out of projection images of line sources by Radon transform. Effect of depth of interaction that gamma photons rip into crystals was modeled when calculating the theoretical position of projection. Several systems were simulated by Monte Carlo method and calibrated both by the proposed method and by using ideal point sources for comparison. Calibration accuracy achieved by the proposed method is comparable to that by using ideal point sources.