A large panel of human Fab fragments against the gp41 subunit of the HIV-1 envelope glycoprotein was isolated by panning six phage-displayed antibody libraries against recombinant gp41. The libraries were prepared from HIV-1-seropositive donors. Twenty-three Fabs recognizing conformation-dependent determinants on gp41 were isolated. Further selection of libraries against (1) gp41 ligated with Fabs from the initial selection and against (2) a recombinant gp41-containing gp140 protein yielded five additional Fabs. Competition of members of the Fab panel with one another and with previously described antibodies revealed a series of overlapping specificities that were conveniently grouped into three major epitope clusters. The majority of Fabs recognized epitopes involving residues 649-668 (previously known as the cluster II region), numbered using the Los Alamos LAI sequence. A second set of Fabs reacted with an epitope involving residues 584-609 (known as the cluster I region). Another set of Fabs appeared to recognize a third conformational epitope that has been termed the cluster III region. This third Fab epitope group demonstrated some overlap with both clusters I and II in binding assays. None of the Fabs neutralized HIV-1 laboratory strains at biologically significant concentrations. This tends to support the opinion that a vaccine based on the gp41 molecule has the drawback that neutralizing epitopes of gp41 are rare and/or unfavorably presented to the immune system. Analysis of heavy chain sequences revealed common CDR3 motif sequences in several antibodies, which appears to be an interesting consequence of a persistent immune response to conserved antigen structures.