A population of 73 donor samples was assembled on the basis of reactive results in routine screening with three different licensed human T-lymphotrophic virus type III (HTLV-III) antibody enzyme-linked immunosorbent assay (ELISA) procedures. The samples were retested by a number of licensed and developmental tests and by Western blot analysis. Our data indicate that nonspecific results are generated by ELISA tests and that many of these reactions appear to be directed against the cell substrate used to grow the virus. These findings suggest that combinations of currently licensed ELISA tests, based upon HTLV-III grown in H-9 cells, cannot be used to confirm the specificity of reactive samples.