Colony stimulating factor-1 (CSF-1) and its receptor (encoded by the c-fms proto-oncogene) have long been recognized as playing an important role in monocytic differentiation. However, the regulation of expression of the CSF-1 and c-fms genes during inhibition of monocytic differentiation has not been fully characterized. The present studies demonstrate that dexamethasone (dex) and cyclosporin A (CsA) resulted in inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of HL60 cells, as well as TPA induction of c-fms and CSF-1 transcripts. These agents also blocked TPA-induced adherence, alpha-naphthyl acetate esterase staining, and the development of a more differentiated morphology. Nuclear run-off analyses revealed no effect of either of these agents on transcription of either c-fms or CSF-1 genes in TPA-treated HL60 cells. Measurements of c-fms transcript half-life confirmed post-transcriptional regulation of c-fms transcript levels after the addition of dex or CsA to TPA, both of which resulted in a decrease in c-fms mRNA half-life. Others have suggested that TPA results in the stabilization of c-fms mRNA in HL60 cells through induction of a labile mRNA stabilizing protein. We observed, however, that the inhibition of protein synthesis by cycloheximide (CH) in this setting of early monocytic differentiation increased both c-fms and CSF-1 steady-state transcript levels. While CH had no effect on the transcription of c-fms and CSF-1 genes in TPA/dex- or TPA/CsA-treated HL60 cells, c-fms mRNA was stabilized after the addition of CH to TPA/dex-treated cells. Taken together, our results suggest the existence of a labile mRNA regulatory protein or proteins, whose actions include destabilization of both c-fms and CSF-1 transcripts after inhibition of TPA-induced monocytic differentiation by dex or CsA.