Infection of monocytes with human immunodeficiency virus type 1_Ba-L (HIV-1_Ba-L ) is significantly inhibited by treatment with the serine protease inhibitor, secretory leukocyte protease inhibitor (SLPI). SLPI does not appear to act on virus directly, but rather the inhibitory activity is most likely due to interaction with the host cell. The current study was initiated to investigate how SLPI interacts with monocytes to inhibit infection. SLPI was found to bind to monocytes with high affinity to a single class of receptor sites (∼7,000 receptors per monocyte, K_D = 3.6 nmol/L). The putative SLPI receptor was identified as a surface protein with a molecular weight of 55 ± 5 kD. A well-characterized function of SLPI is inhibition of neutrophil elastase and cathepsin G. However, two SLPI mutants (or muteins) that contain single amino acid substitutions and exhibit greatly reduced protease inhibitory activity still bound to monocytes and retained anti–HIV-1 activity. SLPI consists of two domains, of which the C-terminal domain contains the protease inhibiting region. However, when tested independently, neither domain had potent anti–HIV-1 activity. SLPI binding neither prevented virus binding to monocytes nor attenuated the infectivity of any virus progeny that escaped inhibition by SLPI. A polymerase chain reaction (PCR)-based assay for newly generated viral DNA demonstrated that SLPI blocks at or before viral DNA synthesis. Therefore, it most likely inhibits a step of viral infection that occurs after virus binding but before reverse transcription. Taken together, the unique antiviral activity of SLPI, which may be independent of its previously characterized antiprotease activity, appears to reside in disruption of the viral infection process soon after virus binding.