ISP-1/myriocin is a new type of remarkably potent immunosuppressant, the structure of which is homologous to sphingosine. ISP-1/myriocin inhibited the proliferation of an IL-2-dependent mouse cytotoxic T cell line, CTLL-2, at nanomole concentrations. ISP-1/myriocin inhibits serine palmitoyltransferase activity at picomole concentrations. This enzyme catalyzes the first step of sphingolipid biosynthesis and reduces the intracellular pool of sphingolipid intermediates. The growth inhibition induced by ISP-1/myriocin was completely abolished by the addition of sphingosines or sphingosine-1-phosphate, but not by sphingomyelin or glycosphingolipids. These results suggest that sphingosines or sphingosine-1-phosphate are associated with CTLL-2 proliferation, and ISP-1/myriocin suppresses T cell proliferation by the modulation of sphingolipid metabolism. ISP-1/myriocin should be a useful tool for the study of the sphingolipid pathway, which has been associated with various kinds of signal transduction.