A number of viruses, when they bind to cells, activate intracellular signals that facilitate post-binding steps of infection. To determine if retroviruses activate intracellular signaling, we transduced HeLa cells with amphotropic retroviruses produced by TelCeB6 cells and examined cell lysates for activated Rac1. We found that retroviruses activate Rac1. Rac1 activation was blocked when cells were depleted of cholesterol, cultured in suspension, or incubated with an anti-beta(1) integrin antibody, and when viruses were treated with heparinase III. Retrovirus activation of Rac1 did not require the amphotropic envelope protein. Gene transfer was reduced 2.4-fold when viruses were treated with heparinase III, but did not change when cells were transduced in the presence of function-blocking anti-beta(1) integrin antibodies. The implications of these findings with respect to retrovirus-cell interactions are discussed.