Strategies that prevent initial HIV infection of cells are greatly needed. In this study, we determined the requirement of divalent cations for HIV infection of and attachment to peripheral blood mononuclear cells (PBMC), which contain several types of HIV-infectable cells-CD4(+) T cells, monocytes and dendritic cells. EDTA, added only during PBMC exposure to HIV, reduced infection by an average of 92%. The reduction of infection by EDTA was accompanied by a reduction in HIV binding to PBMC; R5, X4 and dual-tropic HIV binding to PBMC were inhibited by >85%. EGTA similarly reduced HIV binding to PBMC, while addition of Ca(2+) or Mn(2+), but not Mg(2+), fully restored binding. Virus attachment was inhibited in a dose-dependent manner by trypsin treatment of PBMC, indicating protein involvement in HIV binding. In contrast, mannan or soluble ICAM-1 did not inhibit HIV binding to PBMC. These data indicate that a Ca(2+)-dependent cell-surface protein(s) is responsible for the majority of HIV attachment to and infection of PBMC. Further studies of this are likely to reveal novel strategies to prevent infection of PBMC.