Meiosis is the developmental program by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. When Schizosaccharomyces pombe diploid cells undergo meiosis, they differentiate into asci containing four haploid ascospores that are highly resistant to environmental stress. The formation of the ascospore wall requires the activity of several enzymes involved in the biosynthesis and modification of its components, such as alpha- and beta-glucan synthases. Once the spores are completely mature, the wall of the ascus undergoes an endolytic process that results in the release of ascospores from the ascus, allowing their dispersal into the environment. This process requires the activity of the endo-alpha-1,3-glucanase Agn2. Here, we focus on the characterization of the endo-beta-1,3-glucanase Eng2, which is also required for ascospore release from the ascus. Although Eng2 is present during the mitotic cycle, the protein accumulates after meiosis II. The expression of eng2(+) is required for the efficient release of ascospores, as shown by placing eng2(+) under the control of a repressible promoter. Furthermore, a point mutation that destroys the catalytic activity of the protein results in a phenotype similar to that of the mutant strain. Finally, we demonstrate that exogenous addition of purified Eng2 releases the ascospores from asci generated by an eng2Delta mutant. We propose that Eng2 would act together with Agn2 to completely hydrolyze the ascus wall, thereby assisting in the release of ascospores in S. pombe.