While the cellular sources for granulocyte-macrophage colony-stimulating factor (GM-CSF) are known to be widely distributed among several cell types, interleukin-3 (IL-3) gene expression has been demonstrated in only certain T-cell clones and in blood mononuclear cells stimulated with phytohemagglutinin (PHA) and phorbol-myristate-acetate (PMA). To determine which blood cells were responsible for this expression, we fractionated PHA/PMA-stimulated mononuclear cells and identified T lymphocytes as the source of IL-3 mRNA. Low-level IL-3 expression was detected as well in several stimulated human T-cell lines. Hematopoietic stromal cells such as fibroblasts and endothelial cells could not be induced to express IL-3 mRNA. The kinetics of IL-3 mRNA induction in mononuclear cells and lymphocytes stimulated with PHA/PMA or anti-CD3 monoclonal antibody (MoAb) and interleukin-1 (IL-1) were similar to those observed for GM-CSF expression.