It has been proposed recently that the cell surface peptidase CD26 acts in concert with CD4, the human immunodeficiency virus (HIV) primary receptor molecule, to mediate HIV entry into permissive cells. We have failed to detect significant levels of CD26 cell surface expression and enzymatic activity in a number of commonly propagated human CD4+ cell lines, although CD26 mRNA was present at very low levels, as detected by reverse transcription PCR. No relationship existed between the expression of CD26 and the ability of these cells to be infected with HIV or to fuse to form syncytia. We have tested two inhibitors of CD26 enzymatic activity and several anti-CD26 monoclonal antibodies and found that they inhibit neither HIV infection nor HIV-induced syncytium formation. NIH 3T3 cells stably transfected with the cDNAs for human CD4 and CD26 expressed these molecules at the cell surface and had CD26 enzymatic activity. Inoculation of the double transfectants with HIV did not result in virus entry above the background level, as verified by PCR amplification of viral DNA. We were unable to recover infectious virus from the HIV-inoculated NIH 3T3 double transfectants either by transfer of supernatants or by cocultivation with human CD4+ indicator cells. Moreover, the transfectants did not fuse with HIV-infected cells to form syncytia, nor were syncytia observed in HIV-inoculated cultures. These results are inconsistent with the CD26 molecule being a cofactor for entry of HIV in CD4+ cells.