We have previously described the induction of two sets of sporulation-specific mRNAs in Saccharomyces cerevisiae. Herein we correlate the appearance of these RNAs with the major morphogenic events of sporulation, and we analyze the spatial distribution of the RNAs within the ascus. Several observations suggest that the first set of messages is involved in spore wall synthesis. In fractionation experiments, these mRNAs are detected in the ascal cytoplasm but not in developing spores, indicating that the proteinaceous component of the spore wall is synthesized from the external compartment. The second set of messages is induced later in the course of spore maturation. These mRNAs accumulate within the spores and, unlike the first set of mRNAs, are retained in mature asci until the early stages of germination. We conclude that the development of ascospores proceeds through the differentiation of functionally distinct subcellular compartments.