D-type cyclin-dependent kinase (cdk) activity in mammalian cells has been identified and characterized. When rodent fibroblasts, macrophages, or myeloid cells were lysed with Tween 20 and precipitated with antibodies to cyclins D1, D2, D3, or cdk4, kinase activities in immune complexes were detected that phosphorylated the retinoblastoma protein (pRb) but not histone H1 or casein. These activities were primarily attributed to cdk4 in proliferating cells. When quiescent cells were stimulated to enter the cell cycle, cyclin D1-dependent kinase activity was first detected in mid-G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 lagged behind cyclin induction and correlated with the delayed accumulation of cdk4 and formation of cyclin D1-cdk4 complexes. Thus, cyclin D1-associated kinase activity was not detected during the G0-to-G1 transition. Rodent fibroblasts overexpressing cyclin D1 or cyclin D3 with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Despite continued overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and their kinase activities remained dependent on serum stimulation, indicating that upstream regulators govern the formation of active enzymes. D-type cyclins (D1, D2, D3) are differentially and combinatorially expressed in mammalian cells and are induced earlier in G1 than cyclin E. Cyclin D1 can complement G1 cyclin-deficient yeasts and is a known target of chromosomal translocations in human malignancies. Cyclin D1 can form complexes with cdk2, -4, -5, and -6. Overexpression of cyclin D1 in mammalian fibroblasts shortens G1, reduces cell size, and decreases serum dependency. Conversely, cells microinjected with cyclin D1 antisense vectors or specific antibodies to D1 are inhibited from entering S phase, but injections performed at or after the G1/S transition are without effect. Cyclin D1 is both necessary and rate-limiting for G1 progression in fibroblasts. TGF-β arrests cells in late G1 by inhibiting cdk4 expression and the activity of the cyclin E-cdk2 complex. Overexpression of cdk4 can abrogate TGF-β's ability to arrest cell cycle progression and leads to reactivation of cyclin E-cdk2 histone H1 kinase activity. Cyclin D1-associated kinase activity in immune complexes was detected using specific antibodies and was found to phosphorylate pRb but not histone HD-type cyclin-dependent kinase (cdk) activity in mammalian cells has been identified and characterized. When rodent fibroblasts, macrophages, or myeloid cells were lysed with Tween 20 and precipitated with antibodies to cyclins D1, D2, D3, or cdk4, kinase activities in immune complexes were detected that phosphorylated the retinoblastoma protein (pRb) but not histone H1 or casein. These activities were primarily attributed to cdk4 in proliferating cells. When quiescent cells were stimulated to enter the cell cycle, cyclin D1-dependent kinase activity was first detected in mid-G1, reached a maximum near the G1/S transition, and remained elevated in proliferating cells. The rate of appearance of kinase activity during G1 lagged behind cyclin induction and correlated with the delayed accumulation of cdk4 and formation of cyclin D1-cdk4 complexes. Thus, cyclin D1-associated kinase activity was not detected during the G0-to-G1 transition. Rodent fibroblasts overexpressing cyclin D1 or cyclin D3 with cdk4 exhibited greatly elevated cyclin D-dependent kinase activity, which remained absent in quiescent cells but rose to supraphysiologic levels as cells progressed through G1. Despite continued overproduction of cyclins and cdk4, the assembly of cyclin D-cdk4 complexes and their kinase activities remained dependent on serum stimulation, indicating that upstream regulators govern the formation of active enzymes. D-type cyclins (D1, D2, D3) are differentially and combinatorially expressed in mammalian cells and are induced earlier in G1 than cyclin E. Cyclin D1 can complement G1 cyclin-deficient yeasts and is a known target of chromosomal translocations in human malignancies. Cyclin D1 can form complexes with cdk2, -4, -5, and -6. Overexpression of cyclin D1 in mammalian fibroblasts shortens G1, reduces cell size, and decreases serum dependency. Conversely, cells microinjected with cyclin D1 antisense vectors or specific antibodies to D1 are inhibited from entering S phase, but injections performed at or after the G1/S transition are without effect. Cyclin D1 is both necessary and rate-limiting for G1 progression in fibroblasts. TGF-β arrests cells in late G1 by inhibiting cdk4 expression and the activity of the cyclin E-cdk2 complex. Overexpression of cdk4 can abrogate TGF-β's ability to arrest cell cycle progression and leads to reactivation of cyclin E-cdk2 histone H1 kinase activity. Cyclin D1-associated kinase activity in immune complexes was detected using specific antibodies and was found to phosphorylate pRb but not histone H