Background: Immune cell adaptor protein ADAP (adhesion and degranulation-promoting adaptor protein) mediates aspects of T-cell adhesion and proliferation. Despite this, a connection between ADAP and infection by the HIV-1 (human immunodeficiency virus-1) has not been explored.Results: In this paper, we show for the first time that ADAP and its binding to SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) regulate HIV-1 infection via two distinct mechanisms and co-receptors. siRNA down-regulation of ADAP, or expression of a mutant that is defective in associating to its binding partner SLP-76 (termed M12), inhibited the propagation of HIV-1 in T-cell lines and primary human T-cells. In one step, ADAP and its binding to SLP-76 were needed for the activation of NF-κB and its transcription of the HIV-1 long terminal repeat (LTR) in cooperation with ligation of co-receptor CD28, but not LFA-1. In a second step, the ADAP-SLP-76 module cooperated with LFA-1 to regulate conjugate formation between T-cells and dendritic cells or other T-cells as well as the development of the virological synapse (VS) and viral spread between immune cells.Conclusions: These findings indicate that ADAP regulates two steps of HIV-1 infection cooperatively with two distinct receptors, and as such, serves as a new potential target in the blockade of HIV-1 infection.