Abstract: A new high throughput mutant screening procedure for the detection of sporulation mutants was developed and used to analyse a set of 624 non-lethal homozygous deletion mutants created in the European joint research program EUROFAN. The screening procedure involved determination of LL- and DL-dityrosine, sporulation-specific compounds, which were shown to be robust markers of the extent and arrest stage of sporulation mutants. Secondary screens consisted of light microscopy to detect mature and immature spores and DAPI staining to monitor the progress of meiotic nuclear divisions. We discovered new phenotypic classes of mutants defective in spore wall synthesis that were not discovered by previous screens for sporulation mutants. The genes corresponding to the sporulation mutants fell in several functional classes, some of which were previously unknown to be involved in spore formation. Peroxisomes seem to play a role in spore wall synthesis. Mitochondria play a role in sporulation that is not simply restricted to supply of ATP from respiratory metabolism. The deletion mutants included in the set were functionally unknown at the start of EUROFAN; however, within the last few years the importance to sporulation of some of them was also reported by other authors. Taken together, about 8% of all single gene deletion mutants of non-essential genes of Saccharomyces cerevisiae seem to display a clear and reproducible sporulation phenotype.CI - Copyright 2002 John Wiley & Sons, Ltd.