A novel technique is described which allows the study of the responses of T cell subpopulations stimulated in bulk cultures without interfering with cell-cell interactions. The number and phenotype of lymphoblasts developing following stimulation with phytohaemagglutinin (PHA), anti-CD3, staphylococcal protein A (SPA) and pokeweed mitogen (PWM) was determined in HIV-1- and HIV-1+ patients using a new five-parameter flow cytometric method. We found that normal T cells responded faster to PHA than to any of the other mitogens tested. The peak of the PHA response occurred on day 3, followed by anti-CD3 and SPA on day 4 and PWM mitogen on day 5. Although PHA and anti-CD3 stimulated up to 95% and 80% of lymphocytes, respectively, SPA and PWM stimulated only 40% and 30% of cells, respectively. A defective T cell response was observed in lymphocytes cultured from asymptomatic HIV-1+ patients compared with negative controls. This loss of response was related to a selective mortality of T cells following mitogenic stimulation, referred to as activation-associated lymphocyte death (AALD). The results showed that stronger mitogens (PHA and anti-CD3) induced AALD in a larger proportion (50-60%) of T cells than weaker mitogens such as SPA and PWM (30-40%), and that AALD affected different lymphocyte subsets to different extents. AALD occurred more frequently in total CD8+ and CD45RO+ T cells compared with CD4+ and CD45RA+ T cells, but memory CD4+ T cells were the population most severely affected in samples from HIV-1+ donors.