The T-lymphocyte activation process involves a series of coordinately coupled biochemical events occuring in response to antigen or mitogen. These events have not been completely characterized. The present studies investigate the mechanism of protein synthesis during the initial phase of T-cell activation. Among the early biochemical changes, induction of protein synthesis was observed as early as 10 minutes after mitogen stimulation of T-lymphocytes. This early protein synthesis was inhibited by cycloheximide but was insensitive to actinomycin-D, indicating the presence of preformed mRNA in resting lymphocytes. Since early protein synthesis parallels the increase in protein kinase C activity in activated T-lymphocytes, these two biochemical events may be related. In the present report, amiloride, an inhibitor of Na+/H+ antiport and protein kinase C, significantly inhibited [3H-]leucine and [3H-]thymidine incorporation in a dose-dependent manner into phytohemagglutinin (PHA)-stimulated T-lymphocytes. Furthermore, when Tlymphocytes were stimulated by phorbol myristate acetate, a known activator of protein kinase C, a similar inhibition of protein and DNA synthesis by amiloride was observed. The partially purified cytosol fraction isolated from PHA-activated T-lymphocytes showed a 75 % decrease in protein kinase C-mediated [32P] incorporation from ATP in the presence of 100 pM amiloride. These results suggest that the T-cell activation process following exposure to mitogens involves early protein synthesis, which may be mediated by protein kinase C.