Cell-cell and cell-extracellular matrix contacts influence cellular sensitivity to ionizing radiation. To further define the influence of these interactions on tumor cell survival and cell cycle progression after irradiation without or in combination with the phorbol ester phorbol-12-myristate-13-acetate (PMA), the radiation response of p53 wild-type A549 lung cancer cells grown on polystyrene, fibronectin (FN) or BSA was examined. Confluently growing and log-phase A549 cell cultures irradiated on FN showed significantly greater survival compared to cells irradiated on polystyrene or BSA. There was a significantly greater elevation of G(2)/M cells in FN cultures after irradiation compared to other culture conditions. PMA reduced radiation survival on all three substrata and under both log-phase and confluent culture conditions, but had no effect on the elevation of G(2)/M cells in FN cultures. Induction of Chk1 phosphorylation by irradiation was only seen in FN cultures. Chk2 and Cdk1 phosphorylation and Cdc25C expression also differed between FN and polystyrene cultures. Induction of p53 and p21 by irradiation was modulated but not inhibited by PMA, as were changes in cyclin D1 and pRb. Changes in protein expression and phosphorylation of these cell cycle regulatory proteins coincided tightly with accumulation of cells in G(2)/M after irradiation. These findings clearly demonstrate the influence of both intercellular and cell-substratum interactions on the radiation response without or in combination with PMA and differentiate between the cell survival and cell cycle effects of FN attachment.