We have used both auxotroph and prototroph versions of the latest deletion-mutant library to identify genes required for respiratory growth on solid glycerol medium in fission yeast. This data set complements and enhances our recent study on functional and regulatory aspects of energy metabolism by providing additional proteins that are involved in respiration. Most proteins identified in this mutant screen have not been implicated in respiration in budding yeast. We also provide a protocol to generate a prototrophic mutant library, and data on technical and biological reproducibility of colony-based high-throughput screens.