Myeloperoxidase (MPO) is a major heme enzyme involved in inflammatory responses of polymorphonuclear leukocytes. Using cDNA and intron specific probes for MPO we studied the regulation of MPO expression during myeloid differentiation of the promyelocytic HL-60 leukemia cell line. Mature MPO mRNA species of 3.3, 2.8 and 1.6 kb and heterogeneous nuclear (hn) RNA of greater than 8 and approximately 4 kb were observed in wildtype HL-60 cells. Induction of differentiation of the cells towards either granulocytes or macrophages resulted in a profound decrease (greater than 95%) in the concentration of MPO mRNA levels, showing that gene expression of MPO mRNA is closely linked to the stage of development of myeloid cells. Studies using normal and leukemic hematopoietic cells confirmed these findings and showed that myeloblasts and promyelocytes contain MPO mRNA. Rate of transcription of MPO was measured by a nuclear run-on assay in wild-type and day 3- and day -4 differentiated HL-60 cells and was nearly the same in all three. In contrast, rate of transcription of c-myc in the same nuclei became almost undetectable with induction of differentiation. Overall transcription decreased by 60% and 80% on day 3 and 4 of differentiation, respectively, compared to wild-type cells. Stability of mature MPO mRNA was also measured and found to be the same in wild-type and differentiated HL-60. Half-life of MPO hnRNA was less than or equal to 30 min in wild-type HL-60; nevertheless, this hnRNA was easily detectable 3 days after induction of differentiation of these cells. Taken together, the results show that decreased expression of MPO mRNA with differentiation occurs in part post-transcriptionally, possibly due to a failure in RNA processing. In addition, as overall transcription decreases during differentiation, MPO transcription is concomitantly reduced. This indicates that transcriptional and post-transcriptional mechanisms cooperate in the control of MPO gene expression.