Human saliva contains multiple components that inhibit HIV-1 infection in vitro, which may contribute to low oral HIV-1 transmission. Salivary agglutinin (SAG) is a high-molecular-weight glycoprotein encoded by DMBT-1 and identical to gp340, a member of the lung scavange receptor, cysteine-rich receptor family. gp340 binds to surfactants A and D, which is believed to function in the clearance of microorganisms from the lung, as part of the innate immune response. Previously we reported that SAG (gp340) specifically inhibits HIV-1 infection with broad activity against diverse HIV-1 isolates. This gp340 inhibitory activity is mediated by binding to viral gp120 and involves a region different from the CD4-binding site on gp120. Here, we report that the gp340-binding region is localized to a linear, highly conserved sequence near the stem of the V3 loop that is critical for chemokine receptor interaction during viral binding and infection. The interaction of gp340 with gp120 is enhanced by prebinding of sCD4 to gp120, suggesting that gp340 inhibitory activity is mediated by blocking access of the gp120 to the chemokine receptor.