Prothymosin alpha (ProT alpha) is a nuclear protein related to cell proliferation. Its gene is highly activated during postnatal development at stages containing many proliferating but also differentiating cells. In this report, a study on ProT alpha gene expression during differentiation of human myeloid leukemic (HL-60) cells was undertaken to analyze the possible association of ProT alpha to cell differentiation. When HL-60 cells were induced to differentiate to granulocytes (using retinoic acid) or monocyte/macrophages (using 12-O-tetradecanoylphorbol-13-acetate), a marked down-regulation in the levels of ProT alpha transcript was found. When cell division of immature HL-60 cells was interrupted by either treatment with hydroxyurea or serum starvation, ProT alpha gene expression was not significantly altered. These findings suggest that loss of ProT alpha mRNA in induced HL-60 cells is a differentiation-related event. Examination of the stability of ProT alpha mRNA showed that the stabilization of the ProT alpha transcript is differentially regulated in the two HL-60 lineages. Nuclear run-on experiments revealed that during HL-60 differentiation, the transcriptional activity of the ProT alpha gene does not experience significant variations.