p27(Kip1), an inhibitor of cyclin-dependent kinases, is an important regulator of cell cycle progression. We have previously shown that p27(Kip1) inhibits the G0 to S transition when ectopically expressed in p27-47 mouse fibroblasts arrested at high but not low densities. In the study described here, we identify Gadd45alpha, a member of the growth arrest- and DNA damage-inducible family of proteins, as a potential mediator of the density-dependent effects of p27(Kip1) on cell proliferation. Gadd45alpha mRNA and protein were more abundant in p27-47 cells arrested at high densities than at low densities. Amounts of both decreased and remained low when cells arrested at high densities were exposed to mitogens in the absence, but not in the presence, of ectopically expressed p27(Kip1). Importantly, enforced expression of Gadd45alpha prevented density-arrested mouse fibroblasts from initiating DNA synthesis in response to mitogens. We suggest that amounts of Gadd45alpha above a certain threshold are growth inhibitory and that such amounts are achieved in cells arrested at high but not low densities. For cultures arrested at high densities, the resumption of cell cycle traverse requires a sustained reduction in Gadd45alpha abundance, a process that is induced by mitogens and inhibited by p27(Kip1).