The regulation of DNA (cytosine-5) methyltransferase (DNA MeTase) enzyme activity and gene expression was examined in the monoblastoid U937 cell line induced to differentiate with either dibutyryl cyclic AMP (dbcAMP) or phorbol ester. dbcAMP treatment was found to cause the rapid (< 4 h) suppression of DNA MeTase specific activity, with no DNA MeTase activity detectable after 10 h. Equally, no DNA MeTase activity was detectable in nuclear extracts of fresh peripheral blood monocytes. Using both a U937 DNA MeTase cDNA and a mouse DNA MeTase cDNA as probes, steady-state levels of DNA MeTase mRNA were found to decline sharply between 4 and 15 h after dbcAMP treatment. No DNA MeTase mRNA was detectable after 20 h of dbcAMP treatment. Nuclear run-on analysis showed there to be only a small (40%) suppression of DNA MeTase gene transcription in cells treated with dbcAMP for 24 h, implying a role for post-transcriptional processes in the regulation of DNA MeTase mRNA levels. The observed decline in DNA MeTase activity/mRNA levels appeared to precede the dbcAMP-induced arrest in DNA replication, as judged by the incorporation of tritiated thymidine into DNA. In contrast to the effect of dbcAMP, treatment of U937 cells with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) led to an overall stimulation of DNA MeTase specific activity. The TPA response was found to be complex and broadly consisted of an early (0-15 h) burst of DNA MeTase activity followed by a more gradual sustained increase in DNA MeTase activity after prolonged (16-40 h) TPA treatment. The early phase of high DNA MeTase activity was not mirrored by an increase in steady-state levels of DNA MeTase mRNA, as judged by Northern blot analysis. However, a substantial induction of DNA MeTase mRNA levels was observed after 20-24 h of TPA treatment. Nuclear run-on analysis showed this not to be due to any significant increase in DNA MeTase gene transcription. The observed increases in DNA MeTase activity/mRNA levels were observed whilst cells were undergoing deproliferation. Interestingly, the addition of TPA and more physiological protein kinase C (PKC) activators, such as diacylglycerol and phosphatidylserine, to DNA MeTase-enriched nuclear extracts generated a 4.5-fold and a 1.5-fold increase in DNA MeTase specific activity respectively. The TPA-induced stimulation of DNA MeTase activity could be inhibited by the PKC inhibitor H-9, implicating a role for PKC in the regulation of DNA MeTase activity in vivo.