The time course of the down-regulation of topoisomerase II associated with phorbol-induced differentiation was quantified in two human HL-60 leukemia cell lines. In the line sensitive to phorbol-induced differentiation (S cells), immunoreactive topoisomerase II levels did not begin to fall until 12 hr following exposure of the cells to phorbol ester. Thereafter, levels declined over the next 36 hr. By contrast, phorbol treatment of the phorbol ester tolerant cell line (PET cells) produced a transient decrease in topoisomerase II only to be followed by a recovery to pretreatment levels. The patterns of the alterations in topoisomerase II mRNA levels mirrored those of the alterations in immunoreactive topoisomerase II. Unexpectedly, nuclear run-on studies revealed that transcriptional start sites were not reduced in either cell line following their exposure to phorbol esters. These data suggest that the down-regulation of topoisomerase II is a consequence rather than a cause of phorbol-induced differentiation in HL-60 cells. Furthermore, this down-regulation is not mediated by a decrease in the number of topoisomerase II transcription sites.