Strains of human immunodeficiency virus type 1 (HIV-1) that use the coreceptor CXCR4 (X4 strains) become laboratory adapted (LA) when selected for ability to replicate in leukemic T cell lines such as H9. Compared with patient X4 viruses, the gp120-gp41 complexes of LA viruses have a constellation of common properties including enhanced affinities for CD4, greater sensitivities to inactivations by diverse antibodies and by soluble CD4, increased shedding of gp120, and improved abilities to infect HeLa-CD4 cell clones that contain only trace quantities of CD4. These common characteristics, which may result from a concerted structural rearrangement of the gp120-gp41 complexes, have made it difficult to identify a specific feature that is critical for laboratory adaptation. To test the hypothesis that replication of patient X4 HIV-1 is limited by the low CD4 concentration in H9 cells (7.0 x 10(3) CD4/cell), we constructed H9 derivatives that express at least 10 times more of this receptor. Interestingly, most patient X4 isolates readily grew in these derivative cells, and the resulting virus preparations retained the characteristics of primary viruses throughout multiple passages. In contrast, selection of the same viruses in the parental H9 cells resulted in outgrowth of LA derivatives. We conclude that a weak interaction of patient X4 HIV-1 isolates with CD4 is the primary factor that limits their replication in leukemic T cell lines.