We have previously shown that HIV-1 seropositivity is associated with an increase in the difference between the number of CD3+ lymphocytes and the total number of CD4+ and CD8+ lymphocytes [CD3 - (CD4 + CD8)] among peripheral blood lymphocytes (PBL). To investigate the cellular basis of this increase, PBL from seronegative (SN) and AIDS-free seropositive (SP) homosexual men and intravenous drug users were analyzed by two-color flow cytometry. Results showed that SP compared to SN manifested the expected elevation in calculated [CD3 - (CD4 + CD8)] cells (87 vs 28 cells/mm3; P less than 0.001). Only small differences in lymphocyte populations that could contribute to this increase were observed, namely lymphocytes expressing the CD3+CD4-CD8-phenotype (67 vs 56 cells/mm3; P greater than 0.10) or the CD8dim phenotype (135 vs 142 cells/mm3; P greater than 0.10). However, SP had significantly lower numbers of cells expressing the CD56+CD3- phenotype characteristic of natural killer cells (81 vs 170 cells/mm3; P less than 0.001) and significantly higher numbers of T cells expressing the gamma delta T cell receptor (TCR) (81 vs 62 cells/mm3; P = 0.10). The latter difference was primarily due to higher numbers of cells coexpressing gamma delta-TCR and low levels of CD8 (27 vs 15 cells/mm3; P = 0.009). These data suggest that HIV-1 seropositivity is associated with low numbers of natural killer cells and high numbers of CD8+ gamma delta-TCR lymphocytes. Changes in these populations may reflect altered host defense against HIV-1 or altered T cell kinetics in the presence of HIV-1 infection.