Wee1 tyrosine kinase regulates mitosis by carrying out the inhibitory tyrosine 15 phosphorylation of Cdc2 M-phase inducing kinase. Schizosaccharomyces pombe Wee1 is a large protein, consisting of a C-terminal catalytic domain of approximately 350 amino acids preceded by a N-terminal domain of approximately 550 residues. The functional properties of the Wee1 N-terminal domain were investigated by expressing truncated forms of Wee1 in S. pombe. Both positive and negative regulatory domains were identified. Sequences important for Wee1 function were mapped to a central region (residues 363-408). This region is not required for kinase activity or nuclear localization, suggesting it may be involved in substrate recognition. The negative regulatory domain resides in the N-terminal third of Wee1, Wee1 constructs lacking this domain are more effective at delaying mitosis than wild-type Wee1. The negative regulatory domain contains clusters of potential Cdc2 phosphorylation sites. Investigations to monitor the abundance of Wee1 mRNA and protein during the cell cycle were also carried out.