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Sporulation in the budding yeast Saccharomyces cerevisiae is a developmental program initiated in response to nutritional deprivation. Sps1, a serine/threonine kinase, is required for sporulation, but relatively little is known about the molecular mechanisms through which it regulates this process. Here we show that SPS1 encodes a bona-fide member of the GCKIII subfamily of STE20 kinases, both through phylogenetic analysis of the kinase domain and examination of its C-terminal regulatory domain. Within the regulatory domain, we find Sps1 contains an invariant ExxxPG region conserved from plant to human GCKIIIs that we call the EPG motif; we show this EPG motif is important for SPS1 function. We also find that Sps1 is phosphorylated near its N-terminus on Threonine 12, and that this phosphorylation is required for the efficient production of spores. In Sps1, Threonine 12 lies within a 14-3-3 consensus binding sequence, and we show that the S. cerevisiae 14-3-3 proteins Bmh1 and Bmh2 bind Sps1 in a Threonine 12-dependent fashion. This interaction is significant, as BMH1 and BMH2 are required during sporulation and genetically interact with SPS1 in sporulating cells. Finally, we observe that Sps1, Bmh1 and Bmh2 are present in both the nucleus and cytoplasm during sporulation. We identify a nuclear localization sequence in Sps1 at amino acids 411–415, and show that this sequence is necessary and sufficient for nuclear localization. Taken together, these data identify regions within Sps1 critical for its function and indicate that SPS1 and 14-3-3s act together to promote proper sporulation in S. cerevisiae. [ABSTRACT FROM AUTHOR]