Low circulating CD4 cell numbers and CD4 cell dysfunction are distinguishing features of HIV-mediated disease. The current study delineates the in vivo effects of HIV on distinct functional subsets of CD4 cells in homosexually active men who have been infected with HIV for different lengths of time, and examines the capacity of lymphocytes from these men to proliferate in vitro in response to soluble antigen. Although peripherial blood mononuclear cells from most acquired immune deficiency syndrome (AIDS) patients did not proliferate in response to either tetanus toxoid or Candida albicans, cells from most HIV seropositive men without AIDS, many of whom had been infected for more than 18 mo, responded normally to both. Non-responsiveness in HIV-infected men without AIDS was a late event and was associated with longer duration of infection, lower CD4 cell numbers, and subsequent development of AIDS. A defect in this response was observed in only one of 19 HIV seropositive men whose CD4 levels were greater than 300/mm3, but in eight of 10 with levels less than 300/mm3. The defect could not be attributed to a selective depletion of defined CD4 subpopulations that respond to soluble antigen. Dual-color immunofluorescent flow cytometry indicated that 4B4+, 2H4-, and HB-11- CD4 cells were not lost at a faster rate than other CD4 subsets.