Determinants responsible for HIV-1 infection of T lymphoid cell lines were identified by functional analysis of chimeric proviral clones derived from T-cell line tropic-(HXB2) and non-T-cell line-tropic isolates (YU2, ADA). Replacement of the HXB2 V3 envelope loop sequence with that derived from YU2 resulted in a virus that is no longer T cell line-tropic. However, the reciprocal replacement using HXB2 V3 loop sequences did not confer upon either ADA or YU2 envelope proteins the ability to infect T cell lines. Furthermore, the resultant viruses were incapable of infection of primary lymphocytes. Single, double, and multiple point mutations made within the V3 loop sequence did not result in change in tropism, although mutations involving residue 275 resulted in a virus that was incapable of infecting primary lymphocytes but retained the ability to infect Jurkat T lymphoid cells. These results suggest that the V3 envelope determinant is necessary for T cell line infection, but other determinant(s) in envelope are also necessary to obtain infectious virus expression.