We have isolated cDNA clones encoding human IL-3 from libraries constructed in a modified pcD mammalian expression vector by using mRNA prepared from activated human T cell clones. Amino acid sequence of human IL-3 deduced from DNA sequence of these cDNA clones agrees with that predicted from genomic sequence except at amino acid position 27. Northern blotting analysis and S1 nuclease analysis show that almost all activated T cell clones express IL-3 mRNA with kinetics similar to that observed in mouse T cell clones. However, striking difference was found in the level of granulocyte-macrophage-CSF and IL-3 mRNA expressed in activated human T cells. In contrast to mouse T cell clones, granulocyte-macrophage-CSF mRNA is expressed at least two orders of magnitude more abundant than IL-3 mRNA. Yeast Saccharomyces cerevisiae carrying human IL-3 cDNA fused downstream to alpha-factor leader sequence expressed and secreted biologically active IL-3. Several different rat anti-peptide antisera have been used to confirm the presence of human rIL-3 immunochemically. The immunoreactive human IL-3 expressed in transiently transfected COS7 cells or in yeast was observed to be heterogeneous. Human rIL-3 expressed in COS7 cells has multipotential CSF activity in semisolid cultures of bone marrow cells, and selectively induced the proliferation of My-10+ marrow or cord blood cells in liquid cultures.