The product of the CDC7 gene of Saccharomyces cerevisiae, which is needed for the initiation of mitotic DNA synthesis, has homology with known and putative protein kinases. This homology is confined to the kinase catalytic domain, which has a unique organisation in CDC7. To demonstrate that, nonetheless, CDC7 protein has kinase activity, the gene was subcloned under the control of the SP6 promoter. Protein synthesised by transcription and translation in vitro was capable of transferring 32P from [gamma-32P]ATP to histone. This activity was not dependent on Ca2+ or cyclic nucleotides. A mutation of CDC7 constructed in vitro, in which the organisation of the kinase catalytic domain was converted to that found in all other similar enzymes, was unable to function in vivo, as judged by its inability to complement the cdc7-1 allele. This suggests that the abnormal structure of the CDC7 catalytic domain is a key element in the cellular function of this protein in initiating DNA synthesis.