Primary isolates of human immunodeficiency virus type 1 (HIV-1) were obtained by coculture of peripheral blood lymphocytes (PBLs) from HIV-1-infected people with PBLs from uninfected donors. These viral stocks tend to be resistant to neutralization/inactivation by soluble CD4 (sCD4). When these stocks were passed through the T cell line C8166, virus stocks emerged that were sensitive to sCD4. Pre- and post-C8166 stocks maintained their sCD4-resistant and -sensitive phenotypes, respectively, with further passage in PBLs. Pre- and post-C8166 stocks were biologically cloned by two cycles of limiting dilution. The majority (14 of 17) of pre-C8166 clones were sCD4 resistant, and, conversely, the majority of post-C8166 clones (11 of 12) were sensitive to sCD4. Nucleotide and predicted amino acid sequence analysis in the env (gp120) region revealed a limited number of differences between the clones. The only differences that sorted with biological phenotype were in the first constant (C1) region of gp120. Adaptation to growth in C8166 cells and conversion from the sCD4-resistant to the sCD4-sensitive phenotype represent the emergence to prominence of viral species in the pre-C8166 stock that have a replication advantage in C8166 coincident with increased sensitivity to sCD4.