It has been known since 1986 that CD8 T lymphocytes from certain HIV-1–infected individuals who are immunologically stable secrete a soluble factor, termed CAF, that suppresses HIV-1 replication. However, the identity of CAF remained elusive despite an extensive search. By means of a protein-chip technology, we identified a cluster of proteins that were secreted when CD8 T cells from long-term nonprogressors with HIV-1 infection were stimulated. These proteins were identified as α-defensin 1, 2, and 3 on the basis of specific antibody recognition and amino acid sequencing. CAF activity was eliminated or neutralized by an antibody specific for human α-defensins. Synthetic and purified preparations of α-defensins also inhibited the replication of HIV-1 isolates in vitro. Taken together, our results indicate that α-defensin 1, 2, and 3 collectively account for much of the anti–HIV-1 activity of CAF that is not attributable to β-chemokines.