Cells from the human leukemia cell line HL-60 undergo terminal differentiation when exposed to inducing agents. Differentiation of these cells is always accompanied by withdrawal from the cell cycle. Here we describe the isolation of a cDNA encoding a novel serine protease that is present in HL-60 cells and is downregulated during induced differentiation of these cells. We have named this protease myeloblastin. Down-regulation of myeloblastin mRNA occurs with both monocytic and granulocytic inducers. Myeloblastin mRNA is undetectable in fully differentiated HL-60 cells as well as in human peripheral blood monocytes. We found that regulation of myeloblastin mRNA in HL-60 cells is serum dependent. Inhibition of myeloblastin expression by an antisense oligodeoxynucleotide inhibits proliferation and induces differentiation of promyelocyte-like leukemia cells.