Attenuation coefficient maps (mu-maps) are a useful way to compensate for non-uniform attenuation when performing single photon emission tomography (SPET). A new method was developed to record single photon transmission data and a mu-map for the brain was produced using a four-head SPET scanner. Transmission data were acquired by a gamma camera opposite to a flood radioactive source attached to one of four gamma cameras in the four-head SPET scanner. Attenuation correction was performed using the iterative expectation maximization algorithm and the mu-map. Phantom studies demonstrated that this method could reconstruct the distribution of radioactivity more accurately than conventional methods, even for a severely non-uniform mu-map, and could improve the quality of SPET images. Clinical application to technetium-99m hexamethyl-propylene amine oxime (HMPAO) brain SPET also demonstrated the usefulness of this method. Thus, this method appears to be promising for improvement in the image quality and quantitative accuracy of brain SPET.