Thapsigargin (TG), a sesquiterpene lactone and non‐phorbol 12‐myristate 13‐acetate tumor promoter, stimulates a rapid increase in intracellular free Ca²⁺ ([Ca²⁺]_i) in human T lymphocytes clone P28. The [Ca²⁺]_i response to TG is sustained in the presence of 1 mM extracellular Ca²⁺, while it becomes transient in Ca²⁺‐free medium suggesting that TG activates both the release of Ca²⁺ from intracellular stores and the entry of Ca²⁺ from extracellular spaces. TG‐induced Ca²⁺ influx is completely abolished after cell depolarization caused by increased extracellular concentrations of K⁺. The rise in [Ca²⁺]_i stimulated by TG occurs in the absence of detectable production of inositol phosphates. Moreover, TG does not alter the early biochemical events of T cell activation triggered through the CD2 or the CD3 T cell antigens. Indeed, both inositol phosphate production and intracellular pH increase induced by specific monoclonal antibodies (mAb) remain unchanged after TG treatment. These data suggest that in human T lymphocytes TG releases Ca²⁺ from an intracellular pool by a mechanism which is independent of the phospholipase C metabolic pathway. Preincubation with TG of T cell clone P28 empties both the CD2 and the CD3‐sensitive intracellular Ca²⁺ pool(s). Conversely, prestimulation of T cell clone P28 by CD3 or CD2‐specific mAb inhibits the Ca²⁺‐mobilizing effect of TG. Thus it appears that TG and CD2‐or CD3‐specific mAb mobilize Ca²⁺ from common Ca²⁺ pool(s). Taken together, these results demonstrate that Ca²⁺ influx in human T cells may be linked to mobilization of intracellular Ca²⁺ pools and by a mechanism independent of phosphoinositide hydrolysis. They further indicate that the release of intracellular Ca²⁺ pool(s) may play a major role in the opening of cell membrane Ca²⁺ channels observed during the CD2‐ or CD3‐induced stimulation of human T lymphocytes.