We show that integrin α5 subunit expression is stimulated when human fibrosarcoma HT1080 cells are released from quiescence. The α5 subunit mRNA level in quiescent HT1080 cells was increased 24 hr after their release by 10% fetal bovine serum‐containing medium reaching a maximum of 2.5 fold on day 2. Similar levels of induction of cell‐surface α5 subunit protein as well as b̃1 subunit protein were also observed. This resulted in a significant increase of cell attachment to fibronectin. The serum stimulation also increased α5 subunit promoter activity by twofold which was protein synthesis independent. Subsequent deletion of α5 subunit promoter DNA showed that the cis‐element responsible for the activation is located between ‐ 92 bp and the transcription start site. The promoter activity was not induced until 12 hr after the release. Comparison of the effect of a serum‐free medium and a 10% fetal bovine serum‐supplemented medium revealed that both the DNA synthesis and α5 subunit induction were independent of exogenous growth factors. The increased integrin α5b̃1 appears to function by reducing mitogenic activity since blockade of fibronectin binding to its receptor with a RGD peptide, a monoclonal anti‐fibronectin antibody, or a monoclonal anti‐α5 subunit antibody during the release from quiescence significantly stimulated DNA synthesis. On the other hand, stable overexpression of the α5 subunit resulted in decreased DNA synthesis. © 1995 Wiley‐Liss, Inc.