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The retinoblastoma protein (pRb) inhibits progression through the cell cycle. Although pRb is phosphorylated when G1 cyclin-dependent kinases (Cdks) are active, the mechanisms underlying pRb regulation are unknown. In vitro phosphorylation by cyclin D1/Cdk4 leads to inactivation of pRb in a microinjection-based in vivo cell cycle assay. In contrast, phosphorylation of pRb by Cdk2 or Cdk3 in complexes with A- or E-type cyclins is not sufficient to inactivate pRb function in this assay, despite extensive phosphorylation and conversion to a slowly migrating "hyperphosphorylated form." The differential effects of phosphorylation on pRb function coincide with modification of distinct sets of sites. Serine 795 is phosphorylated efficiently by Cdk4, even in the absence of an intact LXCXE motif in cyclin D, but not by Cdk2 or Cdk3. Mutation of serine 795 to alanine prevents pRb inactivation by Cdk4 phosphorylation in the microinjection assay. This study identifies a residue whose phosphorylation is critical for inactivation of pRb-mediated growth suppression, and it indicates that hyperphosphorylation and inactivation of pRb are not necessarily synonymous.