Ndt80 contributes to the highly regulated cascade of sequential gene expression that directs spore formation in Saccharomyces cerevisiae. This DNA-binding transcriptional activator, which is responsible for the expression of a set of middle sporulation-specific genes, is a target of the meiotic recombination checkpoint. Triggering of this checkpoint prevents phosphorylation and accumulation of active Ndt80. In this study we have investigated the requirements for the activation function of Ndt80 by exploring the role of phosphorylation in the regulation of its activity and by examining the effect of C-terminal truncations. Of three phosphoforms of Ndt80 that we resolved, which we refer to as P approximately Ndt80", P approximately Ndt80', and P approximately Ndt80 in order of increasing electrophoretic mobility, the P approximately Ndt80" and P approximately Ndt80' isoforms correlated with active Ndt80. In particular, P approximately Ndt80" was present in lysates from wild-type sporulating cells and in cells that bypassed checkpoint-mediated arrest as a result of mutations in RAD17, SUM1, or SWE1, or overexpression of NDT80. P approximately Ndt80' was the slowest-migrating isoform that accumulated in Delta ime2/Delta ime2 Delta sum1/Delta sum1 cells in sporulation medium and in mitotic cells that ectopically expressed NDT80. Nonphosphorylated Ndt80 and P approximately Ndt80, which had a slightly lower mobility than nonphosphorylated Ndt80 and was the predominant phosphoform present in checkpoint-arrested cells, correlated with inactive Ndt80. These data are consistent with the notion that extensive phosphorylation, but not Ime2-dependent phosphorylation, of Ndt80 is required for its activity. Examination of the effect of increasingly extensive truncation of the C terminal region of Ndt80 revealed that some functions of Ndt80 were more sensitive to a reduction in its activity than others. In particular, we found that a truncated version of Ndt80 that lacked the last 110 residues was able to promote expression of some middle sporulation-specific genes, but could not direct spore formation. Full activity, however, could be restored to this version of Ndt80 by increasing its level of expression.