The study investigates the activity of D-type cyclin-dependent kinases (CDKs) in mammalian cells, which have not been previously detected. The researchers found that lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins D1, D2, and D3, or to cyclin-dependent kinase 4 (CDK4) yielded kinase activities that phosphorylated the retinoblastoma protein (Rb) but not histone H1 or casein. Cyclin D1-dependent kinase activity in proliferating macrophages and fibroblasts was primarily attributed to CDK4 when quiescent cells were stimulated by growth factors to enter the cell cycle. The kinase activity appeared during the G1 phase, lagged behind cyclin induction, and correlated with the delayed accumulation of CDK4 and formation of cyclin D1-CDK4 complexes. Overexpression of cyclin D1 or cyclin D2 together with CDK4 in rodent fibroblasts resulted in elevated cyclin-dependent kinase activity, but this activity remained absent in quiescent cells and only rose to supraphysiological levels as cells progressed through G1. These findings suggest that upstream regulators govern the formation of active enzymes, despite continued overproduction of cyclins and CDK4. The study also discusses the role of D-type cyclins in governing the progression of mammalian cells through the G1 phase and their interaction with CDKs.The study investigates the activity of D-type cyclin-dependent kinases (CDKs) in mammalian cells, which have not been previously detected. The researchers found that lysis of rodent fibroblasts, mouse macrophages, or myeloid cells with Tween 20 followed by precipitation with antibodies to cyclins D1, D2, and D3, or to cyclin-dependent kinase 4 (CDK4) yielded kinase activities that phosphorylated the retinoblastoma protein (Rb) but not histone H1 or casein. Cyclin D1-dependent kinase activity in proliferating macrophages and fibroblasts was primarily attributed to CDK4 when quiescent cells were stimulated by growth factors to enter the cell cycle. The kinase activity appeared during the G1 phase, lagged behind cyclin induction, and correlated with the delayed accumulation of CDK4 and formation of cyclin D1-CDK4 complexes. Overexpression of cyclin D1 or cyclin D2 together with CDK4 in rodent fibroblasts resulted in elevated cyclin-dependent kinase activity, but this activity remained absent in quiescent cells and only rose to supraphysiological levels as cells progressed through G1. These findings suggest that upstream regulators govern the formation of active enzymes, despite continued overproduction of cyclins and CDK4. The study also discusses the role of D-type cyclins in governing the progression of mammalian cells through the G1 phase and their interaction with CDKs.