Progesterone receptor (PR) function is altered by cell signaling, but the mechanisms of kinase-specific regulation are not well defined. To examine the role of cell signaling in the regulation of PR transcriptional activity, we have utilized a previously developed mammalian-based estrogen-response element promoter array cell model and automated cell imaging and analysis platform to visualize and quantify effects of specific kinases on different mechanistic steps of PR-mediated target gene activation. For these studies, we generated stable estrogen-response element array cell lines expressing inducible chimeric PR that contains a swap of the estrogen receptor-α DNA-binding domain for the DNA-binding domain of PR. We have focused on 2 kinases important for steroid receptor activity: cyclin-dependent kinase 2 and DNA-dependent protein kinase. Treatment with either a Cdk1/2 inhibitor (NU6102) or a DNA-dependent protein kinase inhibitor (NU7441) decreased hormone-mediated chromatin decondensation and transcriptional activity. Further, we observed a quantitative reduction in the hormone-mediated recruitment of select coregulator proteins with NU6102 that is not observed with NU7441. In parallel, we determined the effect of kinase inhibition on hormone-mediated induction of primary and mature transcripts of endogenous genes in T47D breast cancer cells. Treatment with NU6102 was much more effective than NU7441, in inhibiting induction of PR target genes that exhibit a rapid increase in primary transcript expression in response to hormone. Taken together, these results indicate that the 2 kinases regulate PR transcriptional activity by distinct mechanisms.