Attenuation Correction is important among other corrections for quantitative Positron Emission Tomography (PET). A common method is to acquire transmission data using an external source from which attenuation correction factors are derived. The aim of this work was to compare different transmission methodologies for the microPET Focus 220 animal scanner in terms of accuracy, signal-to-noise and scatter. This study included experiments in coincidence mode with and without rod windowing, singles mode with two different energy sources (68Ge and 57Co) and post-injection transmission scanning. In addition, the effectiveness of transmission segmentation was investigated. The propagation of transmission bias and noise into the emission images was also examined. Singles transmission scanning resulted in substantially improved signal-to-noise compared with coincidence measurements. The 57Co measurements provided attenuation coefficients close to the theoretical value for an energy window of 120-125 keV, while the 68Ge single measurements were degraded due to scattering from the object. Transmission scatter correction improved the accuracy for a 10 cm phantom but over-corrected for a mouse phantom. 57Co also resulted in low bias and noise in post-injection transmission scans for emission activities up to 20 MBq. Segmentation worked most reliably for transmission data acquired with 57Co but the minor improvement in accuracy of attenuation coefficients and signal-to-noise did not justify its use, particularly for small subjects. The accuracy and signal-to-noise of activity concentration measurements reflected the accuracy and signal-to-noise of transmission measurements. We conclude that 57Co singles transmission scanning is the most suitable method of attenuation correction on the microPET Focus 220 animal scanner.