Three tectal afferent-fiber systems were experimentally labeled in the cat to learn how their distributions within the superior colliculus were related to the prominent compartments of high acetylcholinesterase activity found in the intermediate gray layer. Presumptive somatic sensory afferents were labeled by injections of horseradish peroxidase-wheatgerm agglutinin conjugate placed at the bulbospinal junction and in the ventral anterior ectosylvian cortex corresponding to somatic sensory area SIV. Vision-related afferents were labeled by injections of the same tracer substance into the lateral suprasylvian visual area. In each animal, a single type of injection was made and a detailed study was carried out to compare the patterns of anterograde labeling and acetylcholinesterase staining in serially adjoining sections through the superior colliculus. Fibers labeled by the three types of injection were distributed in clusters that resembled the acetylcholinesterase-positive patches in the intermediate gray layer. In no case, however, were the afferent-fiber clusters in register with the histochemically defined patches. Instead, the innervations derived from the bulbospinal junction, anterior estosylvian sulcus and lateral suprasylvian visual area all formed patchworks within the acetylcholinesterase-poor domain of the intermediate gray layer. In some instances, the afferent-fiber clusters and enzyme-positive patches appeared to have complementary distributions. In other instances, the afferent-fiber clusters seemed to be arranged in the acetylcholinesterase-poor parts of the intermediate layer in a fashion independent of, but not significantly overlapping, the acetylcholinesterase-positive patches. Not all of the space between the acetylcholinesterase-positive patches was taken up by any one of the afferent-fiber systems labeled. The complementary and non-matching distribution of these afferent systems in relation to the acetylcholinesterase-rich patches of the intermediate gray layer stands in contrast to the spatial registration of two other tectal afferent systems with the zones of high acetylcholinesterase activity. Both nigrotectal and frontotectal afferents converge on the acetylcholinesterase-positive patches. We conclude that afferent systems projecting to the intermediate gray layer can be divided into at least two groups: those innervating the acetylcholinesterase-rich compartments and those avoiding them.(ABSTRACT TRUNCATED AT 400 WORDS)