Induction of differentiation in a variety of model systems is accompanied by cell cycle exit and inhibition of Cdk2 kinase activity. We asked whether inhibition of Cdk2 activity is sufficient to allow differentiation to occur in a non-small cell lung cancer cell line. Treatment of NCI-H358 with flavopiridol, an inhibitor of multiple Cdk's, resulted in growth arrest and induction of mucinous differentiation. The onset of differentiation coincided temporally with loss of Cdk2 kinase activity. Western analysis revealed that flavopiridol treatment resulted in depletion of both cyclin E and D1, suggesting that loss of the regulatory subunits is at least partially responsible for the loss of Cdk kinase activity. Similarly, roscovitine, an inhibitor of Cdk's 1, 2, and 5, but not Cdk4, also induced differentiation in NCI-H358, although the resulting pattern of expression of cell cycle regulatory genes differed from the pattern obtained with flavopiridol. Furthermore, stable expression of an antisense Cdk2 construct in NCI-H358 also resulted in the appearance of a marker of mucinous differentiation. These results show that the inhibition of activity of cyclin dependent kinases, particularly Cdk2, by multiple different mechanisms is accompanied by differentiation. Thus, induction of differentiation is one potential mechanism of action for agents that down-regulate Cdk activity.