The increase of intracellular free calcium concentration ([Ca 2Ο© ] i ) and protein kinase C (PKC) activity are two major early mitogenic signals to initiate proliferation of human peripheral T cells. Bacterial lipopolysaccharide (LPS) is nonmitogenic in human T cells. However, in the presence of monocytes, LPS becomes mitogenic to proliferate T cells. The aim of this study was to define the incompetency of LPS on two mitogenic signals in human peripheral T cells. T cells were isolated from human peripheral blood. [Ca 2Ο© ] i and pH i were determined by loading the cells with the fluorescent dyes, Fura-2 acetoxymethyl ester (Fura-2/AM) and 2Π,7Π-bis(2-carboxyethyl)-5-(and 6)carboxyfluorescein acetoxymethyl ester (BCECF/AM). PKC activity was determined by protein kinase assay and cell proliferation was estimated from the incorporation of [ 3 H]-thymidine. The results indicated that (1) LPS (10 Β΅g/ml) stimulated PKC activity significantly within 5 min, reached a plateau at 30 min, and maintained that level for at least 2 h; and (2) LPS stimulated cytoplasmic alkalinization but did not affect the levels of [Ca 2Ο© ] i and [ 3 H]-thymidine incorporation into T cells. Moreover, the combination of calcium ionophore A23187 with LPS significantly stimulated [ 3 H]-thymidine incorporation into T cells. Thus, the results demonstrate that LPS failed to proliferate T cells, probably because of a lack of the machinery necessary to stimulate the mitogenic signal on [Ca 2Ο© ] i elevation.