Using NMR spectroscopy, we studied purified, human T lymphocytes in a serum-free medium. Purified cells were entrapped inside agarose beads and induced to proliferate by the mitogens phorbol-12-myristate-13-acetate and ionomycin. T lymphocytes in standard culture and inside agarose beads exhibit comparable viability, and similar extent and kinetics of DNA synthesis and interleukin-2 secretion. 31P-NMR revealed decreased phosphomonoester and increased phosphodiester content in cells stimulated for two days or longer. 13C-glucose utilization and 13C-lactate production rates showed that 85% of the utilized glucose was converted to lactate. 1H-NMR spectra of the perfusing media indicated that lactate was also produced from substrates other than glucose or glycogen. Glucose accounted for 25% of the lactate produced by quiescent cells, and for 67% of lactate production by stimulated cells. Glycolysis was enhanced 6-fold within the first 2 hours following stimulation, and 15-fold by 48 or 96 h. Aerobic lactate production was increased 3-fold by 48 h, with only a minor enhancement during the first 12 h of stimulation. Our results indicate a shift from mostly aerobic to mostly anaerobic lactate production in T lymphocytes within the first 90 min of the G0 to G1 transition during cell cycle progression.