Tumor necrosis factor alpha (TNFalpha) potently inhibits the in vitro growth of highly purified human d-6 erythroid colony forming cells (ECFC). Unlike the inhibitory effect of TNFalpha on other cells, including more immature ECFC, this antiproliferative effect of TNFalpha is not related to apoptosis because the d-6 cell descendants were morphologically normal, without apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling assay and without caspase activation by Western blots after TNFalpha treatment. TNFalpha did not appear to affect the cell cycle distribution, but the cell cycle duration was significantly longer in TNFalpha-treated cells. DNA synthesis was also significantly reduced by TNFalpha. Studies of various proteins that regulate the cell cycle showed that cyclin-dependent kinase 6 (CDK6) protein and mRNA levels were concomitantly decreased in the presence of TNFalpha, suggesting that inhibition of cell growth was related to reduced CDK6. To evaluate this, the CDK6 gene was transferred into ECFC using green fluorescence protein-retrovirus-mediated gene transfer. The results showed that the level of cell growth produced by TNFalpha was increased by 30% when the cells were transfected with CDK6. Therefore, the modification of cell cycle progression in the presence of TNFalpha through a reduction of CDK6 is an important mechanism in the TNFalpha inhibition of human ECFC expansion.