In this study we have surveyed by immunoblotting the protein levels of Cyclin D1, D2, D3 and their catalytic partners, Cdk4 and Cdk6 in normal and transformed human cells. We found that all these proteins were differentially expressed in diploid cells derived from different tissues, in contrast to Cyclin E, Cyclin A and Cdk2 which are ubiquitously expressed. D-type Cyclins were never dramatically overexpressed and often very poorly expressed in tumor cell lines when compared to the levels in their normal counterparts. In contrast, Cdk4 was expressed at high levels in several tumor cell lines and Cdk6 was ectopically expressed in two sarcoma lines, suggesting a possible involvement of these two Cdks in oncogenesis. Interestingly, low levels of Cyclin D1 and D3 proteins always correlated with functional inactivation of the retinoblastoma gene product (pRb). In cells displaying active pRb, Cyclin D1 was found associated with Cdk4 regardless of whether the p53 gene was wild-type or mutant. Microinjection during G1 of Cyclin D1 anti-sense cDNA or anti-Cyclin D1 antibody in these cells arrested the cell cycle in G1. In cells lacking pRb function, Cyclin D1 was dissociated from Cdk4. Microinjection during G1 of Cyclin D1 antisense cDNA or anti-Cyclin D1 antibody in these cells did not affect G1 progression. These results show that (i) in the absence of pRb, Cyclin D1 is expressed at low levels, is dissociated from Cdk4 and becomes dispensable in G1; (ii) Cyclin D1 needs to be associated with its catalytic subunit, Cdk4, to function as a positive regulator of G1 progression.