Glutamine, glycine and 10-formyl tetrahydrofolate are consumed during de novo purine biosynthesis. We have found that, in Saccharomyces cerevisiae, synthesis of these cosubstrates is coregulated with synthesis of enzymes of the purine biosynthetic pathway. Analysis of three genes required for synthesis of glutamine, glycine and 10-formyl tetrahydrofolate (GLN1, SHM2 and MTD1, respectively) shows that their expression is repressed by adenine and requires the transcription factors Baslp and Bas2p. Northern analysis reveals that regulation of SHM2 and MTD1 expression by adenine takes place at the transcriptional level. We also show that Bas1p and Bas2p bind in vitro to the promoters of the SHM2 and MTD1 genes, and that mutations in the consensus Bas1p binding sequences strongly affect expression of these genes in vivo. Finally, we have found that a SHM2-lacZ fusion is expressed at a significantly higher level in a bas2-2 disrupted strain than in bas1-2 or bas1-2 bas2-2 mutant strains. The BAS1-dependent, BAS2-independent expression of SHM2-lacZ suggests that, in the absence of Bas2p, Bas1p can interact with another protein partner to activate SHM2 expression.