An accurate, low noise estimate of photon attenuation in the subject is required for quantitative microPET studies of molecular tracer distributions in vivo. In this work, several transmission-based measurement techniques were compared, including coincidence mode with and without rod windowing, singles mode with two different energy sources ((68)Ge and (57)Co), and postinjection transmission scanning. In addition, the effectiveness of transmission segmentation and the propagation of transmission bias and noise into the emission images were examined. The (57)Co singles measurements provided the most accurate attenuation coefficients and superior signal-to-noise ratio, while (68)Ge singles measurements were degraded due to scattering from the object. Scatter correction of (68)Ge transmission data improved the accuracy for a 10 cm phantom but over-corrected for a mouse phantom. (57)Co scanning also resulted in low bias and noise in postinjection transmission scans for emission activities up to 20 MBq. Segmentation worked most reliably for transmission data acquired with (57)Co but the minor improvement in accuracy of attenuation coefficients and signal-to-noise may not justify its use, particularly for small subjects. We conclude that (57)Co singles transmission scanning is the most suitable method for measured attenuation correction on the microPET Focus 220 animal scanner.