Stimulation of T lymphocytes results in the calcium-dependent activation and repression of a large number of genes. However, the functional response made by different T cell subsets is heterogeneous, as their differentiation results in alterations in their sensitivity to activation and in the secretion of cytokines. Here we have investigated the patterns of calcium responses in CD4 and CD8 T cell subsets to help explain their different responses to activation. CD4(+) CD45RA(+) T cells isolated freshly from human blood gave a sustained calcium signal after stimulation, but this was smaller than elicited in CD4(+) CD45RO(+) cells. On in vitro differentiation of CD4(+) CD45RA(+) cells to CD45RO(+), the level of the cytoplasmic calcium response rose initially, but then declined steadily during further rounds of differentiation. The proportion producing an oscillatory calcium response or not responding was increased and differentiation was accompanied by a shift in the calcium between intracellular pools. CD8(+) T cells gave a smaller calcium response than paired CD4(+) T cells and showed a difference in the numbers of cells giving a transient, rather than sustained, calcium signal. The increase in oscillating cells in the CD4(+) CD45RO(+) population may reflect the heterogeneity of this population, particularly in terms of cytokine production. The changing patterns of calcium responses in T cells as they differentiate may explain variation in the cellular response to activation at different stages in their lifespan and emphasize the importance of the both the quantity and the quality of the calcium signal in determining the outcome of T cell activation.