AbstractT lymphocytes may be separated into subsets according to their expression of CD45 isoforms. The CD45R0+T cell subset has been reported to proliferate in response to recall antigen and to mitogenic mAb to a much greater extent than the CD45RA+subset. This difference could be due to more efficient coupling of the T cell antigen receptor complex to mitogenic signaling pathways. To investigate this possibility, CD3 antigen‐induced calcium signals, diacylglycerol (DAG) production and protein kinase C (PKC) activation levels were compared in CD45RA+and CD45R0+human T lymphocyte subsets derived from peripheral blood. The mean CD3‐induced rise in intracellular calcium was 80% greater in CD45R0+than in CD45RA+cells. Basal DAG levels in CD45R0+cells were found to be, on average, 60% higher than in CD45RA+cells (p= 0.002), but the CD3‐induced production of DAG over background was not different in the two subsets(p= 0.4). Basal PKC activity, and CD3‐induced PKC activation levels over background, were found to be 50% and 140% higher, respectively, in CD45R0+cells than in CD45RA+cells (p= 0.015 and 0.023). The CD45R0+subset contained a higher proportion of cells expressing activation markers, such as CD25, CD71 and major histocompatibility complex class II, when compared to the CD45RA+subset. Our results suggest that the elevated basal DAG levels observed in the CD45R0+subset may reflect the recent activation of these cells. Both the higher basal DAG and CD3‐induced elevation in intracellular calcium observed in the CD45R0+cells may contribute to the greater PKC activation signals triggered by CD3 mAb in this subset. These findings elucidate the greater response of CD45R0+T cells to mitogenic stimuli compared to CD4