The dependence upon protein synthesis of physiological and biochemical events occurring during yeast sporulation was investigated. Protein synthesis was inhibited by cycloheximide. There was an early, irreversible sensitivity to inhibition with respect to cell viability and ascus formation; inhibition was reversible only if the cells were inhibited after, but not prior to, 2 to 3 h in sporulation medium. Interruption of protein synthesis of any time during sporulation inhibited all measurable metabolic and sporulation-specific processes except protein breakdown and, to some extent, ribonucleic acid synthesis. The time interval between the occurrence of an event and the protein synthesis necessary for that event was determined to be 2 to 3 h for ascus formation, </=30 min for deoxyribonucleic acid synthesis, 1 h for tetranucleate cell formation, </=30 min for ribonucleic acid breakdown, 1 to 2 h for glycogen synthesis, and 2 to 3 h for glycogen breakdown.