Recent evidence points to the existence of lattices of acetylcholinesterase (AChE) and cytochrome oxidase (CYO) in deep layers of the superior colliculus. We sought to further describe their localization within the rat superior colliculus using new three dimensional (3D) digital imaging techniques. Texture maps of AChE staining within the stratum griseum intermediate (SGI) suggest a sheet of high enzyme activity punctuated by areas of pallor. This lattice of dense staining covered the caudal end of the colliculus and extended rostrally along the medial bank. Discrete regions of low staining were most apparent within the IVc subdivision of SGI. A similar examination of CYO activity failed to reveal any compartmentalization.