Publisher Summary This chapter discusses the preparation of active cdc7/Dbf4 kinase from yeast cells. Saccharomyces cerevisiae cdc7 mutants are blocked in the cell cycle at the G1/S boundary at the restrictive temperature. Biochemical studies have shown that Cdc7 is a serine/threonine protein kinase that phosphorylates histone H1. It has been proposed that Cdc7 kinase is a component, along with Clb5,6/Cdc28 kinase, of S phase-promoting factor (SPF). Thus, there is great interest in determining what activates Cdc7 kinase at G1/S5 and in defining its in vivo substrates. Progress in characterizing the kinase in these respects has been limited, however, not only because it is active only transiently during the cell cycle and is present only at low levels in the cell, but also because it has been unexpectedly difficult to express active Cdc7 in available expression systems and because the only test substrate, histone H1, is not an efficient substrate.